Agents for reversing toxic proteinopathies

ABSTRACT

The present disclosure relates to compositions and methods for the diagnosis and treatment or prevention of proteinopathies, particularly MUC1-associated kidney disease (ADTKD-MUC1 or MKD), Retinitis Pigmentosa (e.g., due to rhodopsin mutations), autosomal dominant tubulo-interstitial kidney disease due to UMOD mutation(s) (ADTKD-UMOD), and other forms of toxic proteinopathies resulting from mutant protein accumulation in the ER or other secretory pathway compartments and/or vesicles, among others. The disclosure also identifies and provides TMED9-binding agents as capable of treating or preventing proteinopathies of the secretory pathway, and further provides methods for identifying additional TMED9-binding agents.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of PCT application No.PCT/US2020/038847, filed Jun. 20, 2020, which claims the benefit of U.S.Provisional Application No. 62/865,096, filed Jun. 21, 2019, entitled“Agents for Reversing Toxic Proteinopathies,” and of U.S. ProvisionalApplication No. 62/878,304, filed Jul. 24, 2019, entitled “Agents forReversing Toxic Proteinopathies.” The entire contents of theaforementioned applications are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to compositions, methods and kits forthe treatment of toxic proteinopathies.

BACKGROUND OF THE INVENTION

Proteinopathies are a class of disease caused by genetic mutations thatresult in protein misfolding, truncation or mutation and theaccumulation of the resulting protein aggregates inside the cell. Thereare no known, effective treatments for toxic proteinopathies. A needtherefore exists for compounds capable of treating toxicproteinopathies, as well as therapeutic methods associated with suchcompounds.

BRIEF SUMMARY OF THE INVENTION

The current disclosure relates, at least in part, to the identificationof a compound, (±)-2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane(identified as “BRD-4780” herein and also previously known as AGN192403), which was discovered herein to be an effective treatment forMUC1-associated kidney disease (MKD) and other toxic proteinopathiesassociated with extended endoplasmic reticulum (ER) residence of certainpolypeptides (e.g., ADTKD-MUC1 or MKD (e.g., due to a frameshift orother mutation in MUC1), Retinitis Pigmentosa (e.g., due to rhodopsinmutations), autosomal dominant tubulo-interstitial kidney disease due toUMOD mutations (ADTKD-UMOD), and other forms of toxic proteinopathiesresulting from mutant protein accumulation in the early secretorypathway, between the ER and cis-Golgi compartments. Such identificationof (±)-2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane as an agentcapable of treating or preventing toxic proteinopathies derives from theinstant disclosure's specific identification of MKD as a toxicproteinopathy caused by a frameshift in MUC1 (“MUC1-fs”) that results inaccumulation of MUC1-fs protein in the early secretory pathway (betweenthe ER and cis-Golgi compartments, in TMED9-enriched vesicles), where itinduces ER stress and activates the unfolded protein response (UPR).

One aspect of the instant disclosure provides a method of treating orpreventing a toxic proteinopathy with(±)-2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane or apharmaceutically acceptable salt thereof.

In one embodiment, the 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptaneis racemic (±) 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane.

In certain embodiments, the2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane is (+)2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane.

In some embodiments, the2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane is (−)2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane.

Optionally, the 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane is ahydrochloride salt of 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane.

Another aspect of the instant disclosure provides a method for treatingor preventing a proteinopathy resulting from mutant protein accumulationin the early secretory pathway in a subject, the method involvingidentifying a subject as having or at risk of developing a proteinopathyresulting from mutant protein accumulation in the early secretorypathway in a subject; and administering2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane or a pharmaceuticallyacceptable salt thereof, to the subject in an amount sufficient to causereduction or improvement of a symptom of the proteinopathy resultingfrom mutant protein accumulation in the early secretory pathway in thesubject, thereby treating or preventing the proteinopathy resulting frommutant protein accumulation in the early secretory pathway in thesubject.

In certain embodiments, the compound causes release of MUC1, UMOD and/orrhodopsin from the early secretory compartment.

In one embodiment, the symptom of the proteinopathy is one or more of:end-stage renal disease, urinalysis revealing minimal protein and noblood, slowly progressive kidney failure, hyperglycemia, gout, a needfor dialysis or kidney transplantation, night blindness; tunnel vision(optionally due to loss of peripheral vision); latticework vision;photopsia (blinking/shimmering lights); photophobia (aversion to brightlights); development of bone spicules in the fundus; slow adjustmentfrom dark to light environments and vice versa; blurring of vision; poorcolor separation; loss of central vision; and/or blindness.

In some embodiments, the subject has a mutation in MUC1, UMOD and/orrhodopsin. Optionally, the MUC1 mutation is a MUC1 frameshift mutation,the UMOD mutation is a C126R UMOD mutation and/or the rhodopsin mutationis a P23H rhodopsin mutation.

In certain embodiments, the pharmaceutical composition that includes2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane is administered to thesubject via the oral route (“per os” or “P.O.”).

In one embodiment, the 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptaneor pharmaceutically acceptable salt thereof further includes apharmaceutically-acceptable carrier/excipient.

Another aspect of the instant disclosure provides a method for reducingor eliminating accumulation of a mutant protein in the ER lumen of acell, in COPI and/or COPII vesicles of a cell, in the cis-Golgi lumen ofa cell, in the medial cisternae of the Golgi of a cell, and/or in thetrans-Golgi network (TGN) of a cell, the method involving administering2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane to the environment ofa cell in an amount sufficient to reduce or eliminate accumulation ofthe mutant protein in the ER lumen of the cell, in COPI and/or COPIIvesicles of a cell, in the cis-Golgi lumen of the cell, in the medialcisternae of the Golgi of the cell, and/or in the trans-Golgi network(TGN) of the cell, thereby reducing or eliminating accumulation of themutant protein in the ER lumen of the cell, in COPI and/or COPIIvesicles of a cell, in the cis-Golgi lumen of the cell, in the medialcisternae of the Golgi of the cell, and/or in the trans-Golgi network(TGN) of the cell.

In one embodiment, the mutant protein is a MUC1 frameshift mutantprotein, a UMOD pathogenic variant or a rhodopsin mutant. Optionally,the MUC1 mutation is a MUC1 frameshift mutation, the UMOD mutation is aC126R UMOD mutation and/or the rhodopsin mutation is a P23H rhodopsinmutation.

An additional aspect of the instant disclosure provides a kit foridentifying a proteinopathy resulting from mutant protein accumulationin the early secretory pathway in a sample, where the kit includes: (a)an oligonucleotide for detection of a MUC1 frameshift mutant, a UMODpathogenic variant and/or a rhodopsin mutant or (b) an antibody(optionally a labeled primary antibody, or the kit may include a labeledsecondary antibody that binds the primary antibody) capable of binding aMUC1 frameshift mutant protein, a UMOD pathogenic variant protein and/ora rhodopsin mutant protein, and instructions for its use.

In certain embodiments, the sample is a sample of a subject having or atrisk of developing a proteinopathy. Optionally, the proteinopathy is aneurodegenerative disease, MUC1-associated kidney disease, autosomaldominant kidney disease caused by uromodulin mutation(s) or a form ofretinitis pigmentosa (RP) caused by a rhodopsin mutation. In certainembodiments, the proteinopathy is MUC1 kidney disease, Uromodulin kidneydisease, Retinitis Pigmentosa, Parkinson's disease and othersynucleinopathies, Familial Danish dementia, CADASIL (cerebral autosomaldominant arteriopathy with subcortical infarcts andleukoencephalopathy), Seipinopathies, Serpinopathies, Type II diabetes,Lysozyme amyloidosis, Dialysis amyloidosis, Cystic Fibrosis, Cataracts,Odontogenic tumor amyloid, Familial British dementia, Hereditarycerebral hemorrhage with amyloidosis (Icelandic), Familial amyloidoticneuropathy or Senile systemic/cardiomyopathy, ApoAII amyloidosis,Familial amyloidosis of the Finnish type (FAF), Fibrinogen amyloidosis,Inclusion body myositis/myopathy, Hereditary lattice corneal dystrophy,Pulmonary alveolar proteinosis or ApoL1-positive kidney disease.

A further aspect of the instant disclosure provides a pharmaceuticalcomposition for treating a subject having or at risk of developing aproteinopathy that includes a therapeutically effective amount of2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane and a pharmaceuticallyacceptable carrier.

An additional aspect of the instant disclosure provides a method forpreparing a 3-isopropylbicyclo[2.2.1]heptan-2-amine salt as a singleenantiomer, the method involving protecting the amino group of a3-isopropylbicyclo[2.2.1]heptan-2-amine salt to form an amide;performing a solvent separation step; and deprotecting the amino group,thereby preparing a 3-isopropylbicyclo[2.2.1]heptan-2-amine salt as asingle enantiomer.

In one embodiment, the protecting group is pNO₂—Cbz. In anotherembodiment, the solvent separation step involves supercritical fluidchromatography, optionally performed on an AD-H column.

Another aspect of the instant disclosure provides a method for preparing2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane as a singleenantiomer, the method involving:

Another aspect of the instant disclosure provides a method of treatingor preventing MUC1-associated kidney disease (MKD) in a subject in needthereof, the method involving administering to the subject2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane or a pharmaceuticallyacceptable salt thereof as a first agent and a second agent that isvitamin D, a phosphate binder, a blood pressure medication or adiuretic, thereby treating or preventing MKD in the subject.

In a further aspect, the instant disclosure provides a method fortreating or preventing a proteinopathy, the method involving contactinga TMED9 gene or gene product with an agent that binds the TMED9 gene orgene product, thereby treating or preventing the proteinopathy.

In one embodiment, the proteinopathy is MUC1-associated kidney disease,autosomal dominant kidney disease caused by uromodulin mutations, a formof retinitis pigmentosa (RP) caused by a rhodopsin mutation, or aneurodegenerative disease (e.g., Alzheimer's disease (AD) or otherdementia; Parkinson's disease (PD) or a PD-related disorder; priondisease (including, e.g., Creutzfeldt-Jakob Disease, variantCreutzfeldt-Jakob Disease, Bovine Spongiform Encephalopathy, Kuru,Gerstmann-Straussler-Scheinker disease, fatal familial insomnia (FFI),scrapie, or other animal TSE); motor neuron disease (MND; including,e.g., Amyotrophic Lateral Sclerosis (ALS), Primary Lateral Sclerosis(PLS), Progressive Bulbar Palsy (PBP), Pseudobulbar Palsy, ProgressiveMuscular Atrophy, Spinal Muscular Atrophy (Type 1, Type 2, Type 3, Type4), or Kennedy's Disease); or spinocerebellar ataxia (SCA)).

In certain embodiments, the proteinopathy is a proteinopathy of thesecretory pathway.

In some embodiments, the agent that binds the TMED9 gene or gene productbinds the TMED9 protein.

In one embodiment, the agent that binds the TMED9 gene or gene productis a small molecule. Optionally, the agent that binds the TMED9 gene orgene product is 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane.

In certain embodiments, the proteinopathy is treated in a cell.

In one embodiment, the proteinopathy is treated in a tissue or subject.Optionally, the subject is human.

In another embodiment, the agent that binds the TMED9 gene or geneproduct inhibits and/or downregulates the TMED9 gene or gene product.

An additional aspect of the instant disclosure provides a method fortreating or preventing a proteinopathy in a cell, the method involvingdisrupting the TMED9 gene of the cell, thereby treating or preventingthe proteinopathy in the cell.

Another aspect of the instant disclosure provides a method for treatingor preventing a proteinopathy in a cell, the method involving contactingthe cell with an agent that binds a TMED9 gene product in the cell,thereby treating or preventing the proteinopathy in the cell.

In one embodiment, the agent that binds the TMED9 gene product inhibitsand/or downregulates the TMED9 gene product.

In certain embodiments, the TMED9 gene product is a TMED9 mRNA.

In another embodiment, the TMED9 gene product is a TMED9 protein.

A further aspect of the instant disclosure provides a method forreleasing a misfolded protein from the secretory pathway of a cell, themethod involving contacting the cell with an agent that binds a TMED9gene or gene product, thereby releasing the misfolded protein from thesecretory pathway of the cell.

In one embodiment, the misfolded protein is a MUC1 mutant protein (e.g.,a MUC1 frameshift mutant protein), a UMOD pathogenic variant protein(e.g., a C126R UMOD mutant protein) and a rhodopsin mutant protein(e.g., a P23H rhodopsin mutant protein).

Another aspect of the instant disclosure provides a method for treatingor preventing a proteinopathy in a subject, the method involvingadministering a pharmaceutical composition that includes an agent thatbinds the TMED9 gene or gene product to the subject, thereby treating orpreventing the proteinopathy in the subject.

In one embodiment, the subject is human.

An additional aspect of the instant disclosure provides a method foridentifying a candidate TMED9 gene- or gene product-binding agent, themethod involving (a) providing a cell harboring a misfolded protein ofthe secretory pathway; (b) contacting the cell with a test compound; and(c) identifying removal of the misfolded protein from the cell in thepresence of the test compound, as compared to an appropriate control,thereby identifying the test compound as a candidate TMED9 gene- or geneproduct-binding agent.

In certain embodiments, the test compound is a small molecule.

In other embodiments, the test compound is a macromolecule. Optionally,the test compound is a nucleic acid.

In one embodiment, step (c) includes identifying preferential removal ofthe misfolded protein from the cell, as compared to the correspondingwild-type form of the misfolded protein.

In certain embodiments, the removal of the misfolded protein from thecell is detected as a change in fluorescence. Optionally, the removal ofthe misfolded protein from the cell is detected as a reduction inimmunofluorescence.

In some embodiments, the misfolded protein is labeled.

A further aspect of the instant disclosure provides a method fortreating or preventing a proteinopathy, the method involving contactinga TMED9 gene or gene product with an agent that binds the TMED9 gene orgene product, where the agent that binds the TMED9 gene or gene productis identified by: (a) providing a cell harboring a misfolded protein ofthe secretory pathway; (b) contacting the cell with a test compound; and(c) identifying the test compound as an agent that binds the TMED9 geneor gene product by detecting removal of the misfolded protein from thecell in the presence of the test compound, as compared to an appropriatecontrol, thereby treating or preventing the proteinopathy.

Another aspect of the instant disclosure provides a pharmaceuticalcomposition for treating a subject having or at risk of developing aproteinopathy of the secretory pathway, the pharmaceutical compositionincluding a therapeutically effective amount of a TMED9 gene- or geneproduct-binding agent and a pharmaceutically acceptable carrier.

In certain embodiments, the proteinopathy of the secretory pathway isMUC1-associated kidney disease, autosomal dominant kidney disease causedby uromodulin mutations, a form of retinitis pigmentosa (RP) caused by arhodopsin mutation, or a neurodegenerative disease (e.g., Alzheimer'sdisease (AD) or other dementia; Parkinson's disease (PD) or a PD-relateddisorder; prion disease (including, e.g., Creutzfeldt-Jakob Disease,variant Creutzfeldt-Jakob Disease, Bovine Spongiform Encephalopathy,Kuru, Gerstmann-Straussler-Scheinker disease, fatal familial insomnia(FFI), scrapie, or other animal TSE); motor neuron disease (MND;including, e.g., Amyotrophic Lateral Sclerosis (ALS), Primary LateralSclerosis (PLS), Progressive Bulbar Palsy (PBP), Pseudobulbar Palsy,Progressive Muscular Atrophy, Spinal Muscular Atrophy (Type 1, Type 2,Type 3, Type 4), or Kennedy's Disease); or spinocerebellar ataxia(SCA)).

In some embodiments, the TMED9 gene- or gene product-binding agentdisrupts, inhibits and/or downregulates the TMED9 gene or gene product.

Definitions

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. “About” canbe understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value.

In certain embodiments, the term “approximately” or “about” refers to arange of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less ineither direction (greater than or less than) of the stated referencevalue unless otherwise stated or otherwise evident from the context(except where such number would exceed 100% of a possible value).

Unless otherwise clear from context, all numerical values providedherein are modified by the term “about.”

The term “administration” refers to introducing a substance into asubject. In general, any route of administration may be utilizedincluding, for example, parenteral (e.g., intravenous), oral, topical,subcutaneous, peritoneal, intraarterial, inhalation, vaginal, rectal,nasal, introduction into the cerebrospinal fluid, or instillation intobody compartments. In some embodiments, administration is oral.Additionally or alternatively, in some embodiments, administration isparenteral. In some embodiments, administration is intravenous.

By “agent” is meant any small compound (e.g., small molecule), antibody,nucleic acid molecule, or polypeptide, or fragments thereof or cellulartherapeutics such as allogeneic transplantation and/or CART-celltherapy.

A proteinopathy is a disease, disorder, or dysfunction in which abnormalprotein metabolism or accumulation has been implicated. Someproteinopathies may include neurodegenerative diseases, cognitiveimpairment, lysosomal storage diseases, immunologic diseases,mitochondrial diseases, ocular diseases, inflammatory diseases,cardiovascular diseases, and proliferative diseases, etc. Further,included under the umbrella definition of proteinopathies are suchspecific pathologies as synucleinopathies, tauopathies, amyloidopathies,TDP-43 proteinopathies and others.

By “control” or “reference” is meant a standard of comparison. In oneaspect, as used herein, “changed as compared to a control” sample orsubject is understood as having a level that is statistically differentthan a sample from a normal, untreated, or control sample. Controlsamples include, for example, cells in culture, one or more laboratorytest animals, or one or more human subjects. Methods to select and testcontrol samples are within the ability of those in the art.Determination of statistical significance is within the ability of thoseskilled in the art, e.g., the number of standard deviations from themean that constitute a positive result.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. “Isolate” denotes a degree ofseparation from original source or surroundings. “Purify” denotes adegree of separation that is higher than isolation.

As used herein, the term “subject” includes humans and mammals (e.g.,mice, rats, pigs, cats, dogs, and horses). In many embodiments, subjectsare mammals, particularly primates, especially humans. In someembodiments, subjects are livestock such as cattle, sheep, goats, cows,swine, and the like; poultry such as chickens, ducks, geese, turkeys,and the like; and domesticated animals particularly pets such as dogsand cats. In some embodiments (e.g., particularly in research contexts)subject mammals will be, for example, rodents (e.g., mice, rats,hamsters), rabbits, primates, or swine such as inbred pigs and the like.

As used herein, the terms “treatment,” “treating,” “treat” and the like,refer to obtaining a desired pharmacologic and/or physiologic effect.The effect can be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or can be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease or condition in a mammal, particularly in ahuman, and includes: (a) preventing the disease from occurring in asubject which can be predisposed to the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease, i.e., arresting itsdevelopment; and (c) relieving the disease, i.e., causing regression ofthe disease.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

The phrase “pharmaceutically acceptable carrier” is art recognized andincludes a pharmaceutically acceptable material, composition or vehicle,suitable for administering compounds of the present disclosure tomammals. The carriers include liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject agent from one organ, or portion of the body,to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; and other non-toxiccompatible substances employed in pharmaceutical formulations.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it is understood thatthe particular value forms another aspect. It is further understood thatthe endpoints of each of the ranges are significant both in relation tothe other endpoint, and independently of the other endpoint. It is alsounderstood that there are a number of values disclosed herein, and thateach value is also herein disclosed as “about” that particular value inaddition to the value itself. It is also understood that throughout theapplication, data are provided in a number of different formats and thatthis data represent endpoints and starting points and ranges for anycombination of the data points. For example, if a particular data point“10” and a particular data point “15” are disclosed, it is understoodthat greater than, greater than or equal to, less than, less than orequal to, and equal to 10 and 15 are considered disclosed as well asbetween 10 and 15. It is also understood that each unit between twoparticular units are also disclosed. For example, if 10 and 15 aredisclosed, then 11, 12, 13, and 14 are also disclosed.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 aswell as all intervening decimal values between the aforementionedintegers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,and 1.9. With respect to sub-ranges, “nested sub-ranges” that extendfrom either end point of the range are specifically contemplated. Forexample, a nested sub-range of an exemplary range of 1 to 50 maycomprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

The term “pharmaceutically acceptable salts, esters, amides, andprodrugs” as used herein refers to those carboxylate salts, amino acidaddition salts, esters, amides, and prodrugs of the compounds of thepresent disclosure which are, within the scope of sound medic judgment,suitable for use in contact with the tissues of patients without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use,as well as the zwitterionic forms, where possible, of the compounds ofthe disclosure.

The term “salts” refers to the relatively non-toxic, inorganic andorganic acid addition salts of compounds of the present disclosure.These salts can be prepared in situ during the final isolation andpurification of the compounds or by separately reacting the purifiedcompound in its free base form with a suitable organic or inorganic acidand isolating the salt thus formed. Representative salts include thehydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate,oxalate, valerate, oleate, palmitate, stearate, laurate, borate,benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionateand laurylsulphonate salts, and the like. These may include cationsbased on the alkali and alkaline earth metals, such as sodium, lithium,potassium, calcium, magnesium, and the like, as well as non-toxicammonium, tetramethylammonium, tetramethylammonium, methlyamine,dimethlyamine, trimethlyamine, triethlyamine, ethylamine, and the like.(See, for example, S. M. Barge et al., “Pharmaceutical Salts,” J. Pharm.Sci., 1977, 66:1-19 which is incorporated herein by reference.).

A “therapeutically effective amount” of an agent described herein is anamount sufficient to provide a therapeutic benefit in the treatment of acondition or to delay or minimize one or more symptoms associated withthe condition. A therapeutically effective amount of an agent means anamount of therapeutic agent, alone or in combination with othertherapies, which provides a therapeutic benefit in the treatment of thecondition. The term “therapeutically effective amount” can encompass anamount that improves overall therapy, reduces or avoids symptoms, signs,or causes of the condition, and/or enhances the therapeutic efficacy ofanother therapeutic agent.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention.

Other features and advantages of the disclosure will be apparent fromthe following description of the preferred embodiments thereof, and fromthe claims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this disclosure belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present disclosure,suitable methods and materials are described below. All publishedforeign patents and patent applications cited herein are incorporatedherein by reference. All other published references, documents,manuscripts and scientific literature cited herein are incorporatedherein by reference. In the case of conflict, the present specification,including definitions, will control. In addition, the materials,methods, and examples are illustrative only and not intended to belimiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the disclosure solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings, in which:

FIGS. 1A to 1G demonstrate that mutant MUC1-fs is retainedintracellularly in tubular epithelial cells. FIG. 1A shows periodicacid-schiff (PAS) staining of kidney biopsies from a normal individual(left panel) and an MKD patient (right panel), with the latter showingprominent dilated tubules. FIG. 1B shows immunoperoxidase staining forMUC1-wt protein (apical tubular cell staining, left) and MUC1-fs protein(diffuse intracellular tubular cell staining, right) in an MKD patientkidney biopsy. FIG. 1C shows PAS-stained kidney sections from 24 monthold +/+ and wt/+ female knock-in mice, serving as a negative control.(n=67+/+ mice; 23 wt/+ mice). FIG. 1D shows immunoperoxidase stainingfor MUC1-wt protein (apical tubular cell staining, left) and MUC1-fsprotein (diffuse intracellular tubular cell staining, right) from a fs/+knock-in mouse showed similar localization as in human MKD kidney tissue(FIG. 1B). FIG. 1E shows PAS stained kidney sections from female fs/+knock-in mice at ages 4, 8, 12 and 24 months old, illustratingprogressively prominent dilated tubules as the disease advances. Kidneyregions (cortex, medulla) are marked with dashed lines. (n=23 fs/+mice). FIG. 1F shows immunofluorescence (IF) co-staining of distaltubule in MKD patient kidney organoid for MUC1-wt (red), MUC1-fs(green), E-cadherin (blue) and Na+/K+ ATPase (yellow), which showed thatMUC1-fs was localized intracellularly (middle) compared to MUC1-wt,which was apical (left). FIG. 1G depicts IF co-staining in P cells forMUC1-fs (green), MUC1-wt (red) and Hoechst (grey), which showed thatMUC1-fs was localized intracellularly (middle) compared to MUC1-wt,which was found on the plasma membrane (left).

FIGS. 2A to 2H show that MUC1-fs accumulation triggers the ATF6 branchof the UPR. FIG. 2A shows UPR branch activation analysis in N and Pcells. Generalized (Complex), or specific branch activation (ATF6, PERKand IRE1) was evaluated using a previously described UPR branchtranscriptome (Adamson et al., 2016). Z-scores of normalized expressionvalues obtained from RNA-Seq were used to generate scaled meanexpression profiles. A single boxplot represents the genes in theindicated UPR arm (n=3 replicates). FIG. 2B shows that inhibition of thethree UPR branches demonstrates a specific involvement of ATF6 in P cellcytoprotection. Cell apoptosis was calculated using the protocolsummarized in FIG. 9G below, after 72 hour treatment of N and P cellswith either PERK inhibitor (PERKi, GSK2656157, 10 μM), IRE inhibitor(IREi, 4μ8c, 10 μM) or ATF6 inhibitor (ATF6i, PF-429242, 10 μM). Caspase3/7 activation was calculated as the fraction of caspase 3/7 positivecells. Values are means+SD. (n=4 replicates). FIG. 2C depicts immunoblotand RT-PCR analysis of downstream effectors of the three UPR branches inN and P cells. Protein abundance of BiP (Complex), GRP94 and ERp72(ATF6), ATF4 and CHOP (PERK) and mRNA levels of spliced XBP1 (sXBP1;IRE) were consistent with trends in transcriptomic data in FIG. 2A andcell protection data in FIG. 2B. (n>3 replicates). FIG. 2D showsactivation of downstream effectors (as described in FIG. 2C) of all UPRbranches, including PERK, in P compared to N cells after treatment withTHP (100 nM) for 12 hours. (n>3 replicates). FIG. 2E shows lowmagnification IF images of kidney section stained for MUC1-wt (blue),MUC1-fs (green) and ERp72 (red) in 4 month old female fs/+ mice (beforedisease onset). Images demonstrate co-staining of MUC1-fs with the ATF6effector ERp72 in the tubules of the outer medulla. FIG. 2F showsresults of immunoblot analysis of MUC1-fs as well as downstreameffectors of ATF6 and PERK branches in 12 month old female+/+ and fs/+mice (advanced disease). Increased abundance of calreticulin, ERp72 andGRP94 (ATF6 branch), and CHOP (PERK branch) was observed in fs/+ mice.(n=3 mice/genotype). FIG. 2G is a digital illustration of representativekidney sections stained with TUNEL to detect apoptotic cells in 12 monthold female+/+ and fs/+ mice (advanced disease). All cells detected inthe tissue section have been plotted according to their slide position(grey dots). Increased number of TUNEL positive cells (red dots) isshown in fs/+ kidney section mainly in the area of tubular damage(dashed line). FIG. 2H presents a graph depicting increased apoptosis in12 month old female fs/+ mice versus female+/+ mice, as indicated byTUNEL staining quantification. Values are means+SD (n=3 mice/genotype).See also FIGS. 9A-9J below.

FIGS. 3A to 3J depict that BRD-4780 clears mutant MUC1-fs. FIG. 3A showsa schematic illustration demonstrating the high content screen (HCS)strategy that resulted in the identification of BRD-4780. FIG. 3B showsprimary screen results showing IF quantification of MUC1-fs versus MUC1fs/wt ratio in P cells treated for 48 hours with each of 3713 compoundsof the Repurposing Library at 5 dose points. DMSO (light yellow) was thenegative control. JQ1 (orange) was used as a positive control. Allcompounds are marked in gray and progressively higher concentrations ofBRD-4780 are highlighted with a pink to red dot color gradient (also inFIGS. 13A-13H). FIG. 3C shows secondary screen results demonstrating IFquantification of MUC1-fs versus MUC1 fs/wt ratio in P cells treated for48 hours with each of 203 compounds (identified as hits from the primaryscreen) at 10 dose points. DMSO (light yellow) was the negative control.JQ1 (orange) was used as a positive control. FIG. 3D depicts results ofthe MUC1-fs profiling assay showing IF quantification of MUC1-fs versusMUC1-wt in P cells treated for 48 hours at 10 dose points with each of71 compounds derived from the secondary screen. FIG. 3E depicts resultsof the MUC1 transcription assay showing measurement of total MUC1 mRNAversus IF quantification of MUC1 fs/wt ratio in P cells treated for 24hours and 48 hours, respectively, with 71 compounds from the secondaryscreen. Only doses active in removing MUC1-fs are shown. FIG. 3F depictsresults of a cell viability assay showing quantification for P cellstreated for 5 days with 71 compounds from the secondary screen in theabsence versus presence of THP (100 nM). Only doses active in removingMUC1-fs are shown. Cell viability was measured using live imaging,followed by measuring the fraction of live cells (cells negative tocaspase 3/7 and DRAQ7). FIG. 3G presents IF images of P cells treatedfor 48 hours with DMSO or BRD-4780 (5 μM). MUC1-wt (yellow), MUC1-fs(green), Hoechst (grey). FIG. 3H shows BRD-4780 dose-response curves forIF-detected MUC1-fs, MUC1-wt and cell number in P cells treated as inFIG. 3G. Solid lines represent the best fits of the data to thefour-parameter dose-response curve (GraphPad Prism software). EC50=143nM. Values are means±SD. FIG. 3I comprises bright field images ofrepresentative P cells pre-treated for 48 hours with BRD-4780 (5 μM) orDMSO followed by 5 days co-treatment with BRD-4780 (5 μM) (or DMSO) plusTHP (100 nM). Dead cells were identified based on far redautofluorescence. FIG. 3J shows dose response curves of P cell viabilityupon THP treatment as in FIG. 3I. Cell viability was measured as livecell number (See Example 1 below). Solid lines represent the best fitsof the data to the four-parameter dose-response curve (GraphPad Prismsoftware). EC₅₀ [THP]=17 nM; EC₅₀ [THP+BRD-4780]=75 nM. Values aremeans±SD. (n=3 replicates). See also FIGS. 10A and 10B below.

FIGS. 4A to 4G show that BRD-4780 removed mutant MUC1-fs from kidneys ofheterozygous knock-in mice and human iPSC-derived kidney organoids. FIG.4A shows IF images of MUC1-fs (green), MUC1-wt (red) and NCC (blue) infs/+ mice treated for 7 days with vehicle (left) or BRD-4780 (50 mg/kg,middle) compared with vehicle treated+/+ mice (right). FIG. 4B presentsresults of mean MUC1-fs IF intensity in NCC-positive cells in kidneysections from fs/+ and +/+ mice treated with vehicle or BRD-4780 (1, 10and 50 mg/kg) for 7 days. Mean intensity values were normalized tovehicle treated+1+ mice (0%) and to vehicle treated fs/+ mice (100%).Values are means f SD. (n=4 mice/genotype/dose; see Example 1 below fordetails). FIG. 4C shows immunoblot analysis of MUC1-fs in kidney lysatesfrom fs/+ mice treated with vehicle or BRD-4780 (1, 10 and 50 mg/kg) for7 days. (n=4 mice/genotype/dose). FIG. 4D shows results of RNA-Seqanalysis of mouse kidney lysates from fs/+ mice treated with BRD-4780(50 mg/kg/day) or vehicle for 7 days revealed GO pathways downregulatedby BRD-4780. (n=3 mice/genotype). FIG. 4E shows IF images of MUC1-wt(red), MUC1-fs (green) and laminin (blue) in representative iPSC-derivedkidney organoids from a normal individual (N1) and from three MKDpatients (P1, P2 and P3), each treated for 72 hours with DMSO orBRD-4780 (10 μM). FIG. 4F depicts reduction in MUC1-fs protein abundancein human iPSC-derived kidney organoids generated from three MKD patientsafter treatment with BRD-4780 for 72 hours. Mean fluorescence intensityof tubular MUC1-fs was calculated using the protocol summarized in FIG.12B below. Mean intensity values were normalized to DMSO treated normalorganoids (0%) and to DMSO treated patient organoids (100%). Values aremeans±SD. (n=3 replicates). FIG. 4G shows results that depict that therewas no effect of BRD-4780 treatment on MUC1-wt abundance in humaniPSC-derived kidney organoids generated as in FIG. 4F. Mean intensityvalues were normalized to DMSO treated normal organoids (100%). Valuesare means±SD (n=3). See also FIGS. 12A and 12B below.

FIGS. 5A to 5E show that MUC1-fs accumulated in the early secretorypathway, in a TMED9-positive compartment. FIG. 5A is a schematic of themammalian cell secretory pathway including relevant cellularcompartments involved in vesicular transport, color code as labeled.FIG. 5B shows a subcellular distribution of MUC1-fs in P cells asdetected by MUC1-fs co-localization with organelle-specific markerscalnexin (Canx, ER), SEC31A (COPII), ERGIC-53 (ERGIC), TMED9 (COPI),GM130 (cis-Golgi), TGN46, (trans-Golgi), EEA1 (early endosomes), Rab7(late endosomes) and LAMP1 (lysosomes). Co-localization was calculatedusing the protocol summarized in FIG. 13A below. Representative imagesof MUC1-fs colocalization with the organelle-specific markers are shownin FIG. 13B below. Values are means±SD. (n=3 replicates). FIG. 5Cpresents representative IF images showing co-localization of MUC1-fs(green) with TMED9 (red) in four systems: P cells, fs/+ mouse kidneysections, MKD patient iPSC-derived kidney organoids and kidney sectionof MKD patient. DAPI (grey). FIG. 5D depicts changes observed in thesubcellular distribution of MUC1-fs in P cells after 3 and 5 hours ofBRD-4780 treatment (5 μM). Changes are shown as the percentages of DMSOcontrol (as in FIG. 5B). (n=3 replicates). FIG. 5E shows immunoblotanalysis of MUC1-fs in P cells following 24 hour inhibition ofanterograde ER-Golgi transport by BFA (200 ng/mL) or inhibition oflysosomal degradation by Bafilomycin A (100 nM) in the absence orpresence of BRD-4780 (5 μM). Both perturbations abolished the BRD-4780effect (n=3 replicates). See also FIGS. 13A-13D below.

FIGS. 6A to 6F show the mechanism of action of BRD-4780 by engagement ofits target, TMED9. FIG. 6A presents IF images of MUC1-fs (green), TMED9(red) and MUC1-wt (blue) showing increased abundance of TMED9 inMUC1-fs-positive tubules in patient iPSC-derived kidney organoids(middle) compared to normal control (top). TMED9 increased abundance wasdirectly correlated with the increase of MUC1-fs and was reversed byBRD-4780 treatment (10 μM; 72 hours). FIG. 6B shows immunoblot analysisof P cells in which TMED9 was knocked out using CRISPR-Cas9 with twodifferent sgRNAs (KO1 and KO2). Non-targeting sgRNAs were used ascontrols (NTC1 and NTC2). BRD-4780 (5 μM) treatment was applied for 72hours and the abundance of TMED9, MUC1-fs and the coatomer protein13-COP was tested. Knock out of TMED9 phenocopied the BRD-4780 effect.FIG. 6C presents IF images of MUC1-fs (green) and Hoechst (grey) in Pcells after TMED9 or Nischarin deletion compared to cells treated withnon-targeting sgRNA control (NTC) before and after treatment withBRD-4780 (5 μM) for 72 hours. FIG. 6D shows mean MUC1-fs IF intensity inP cells treated as in FIG. 6C. TMED9 deletion (red) phenocopied theBRD-4780 effect. Values are means±SD. (n>3 replicates). FIG. 6E depictsincreased thermal stability assessed by CETSA suggestive of directbinding of BRD-4780 to TMED9. Representative immunoblot (top) anddensitometric analysis (bottom) of TMED9 abundance in P cells atescalating temperatures (as indicated) with or without treatment withBRD-4780 (5 μM; 1 hour). Higher abundance of TMED9 was noted attemperatures>47° C. in the presence of BRD-4780. Solid lines representthe best fit of the data to the Boltzmann sigmoid. Values are means±SEM.(n=3 replicates). FIG. 6F depicts a schematic of a proposed BRD-4780mechanism of action illustrating the untreated tubular epithelial cell(top) with MUC1-fs trapped in the early secretory pathway (inTMED9-enriched compartments). Without wishing to be bound by theory,following either engagement of TMED9 by BRD-4780 or TMED9 deletion(bottom), MUC1-fs is released from COPI/cis-Golgi/COPII compartments,thus allowing its anterograde transport through the secretory pathway,ultimately resulting in lysosomal degradation.

FIGS. 7A to 7F show the generation and characterization of a MUC1-fsknock-in fs/+ mouse. FIG. 7A is a schematic of hMUC1-wt and hMUC1-fsgenomic constructs (the latter differing only by a single +C (cytosine)insertion (red asterisk) in exon 2) used to generate the wt/+ and fs/+knock-in mouse models. Box (grey dotted line) shows extent of knock-infs human gene sequence. FIG. 7B presents schematics of hMUC1-wt (left)and hMUC1-fs (right) proteins encoded by the mouse knock-in transgenetranscripts. SP, signal peptide. VNTR, variable number of tandemrepeats. SEA, sperm protein enterokinase and agrin domain. TMD, transmembrane domain; fs-VNTR, mutant neosequence VNTR; Neopeptide, uniqueneosequence C-terminal to the FS-VNTR. FIG. 7C shows immunoblot analysisof MUC1-wt and MUC1-fs expression in whole kidney lysates from +1+ andfs/+KI mice. (n=4 replicates). FIG. 7D shows immunoperoxidase stainingfor MUC1-wt (top) and MUC1-fs (bottom) in kidney sections from +/+ andfs/+KI mice. FIG. 7E depicts serum creatinine levels in +/+, wt/+ andfs/+ mice as function of age and gender. Values are means±SEM. *annotates statistically significant difference between fs/+ females +/+females (for number of animals tested, please refer to FIG. 17 below).FIG. 7F shows PAS-stained kidney sections from male fs/+ mice at ages 4,8, 12 and 24 months, illustrating disease progression (n=20 mice).

FIGS. 8A and 8B show MUC1-fs and MUC1-wt distribution in differentsegments of kidneys of fs/+ mouse. FIG. 8A presents IF images thatdemonstrate the distribution of MUC1-wt (red) and MUC1-fs (green) inkidneys of 4 month old female fs/+ mice. Top, low magnification of anentire kidney section, showing the location of the subsequent highmagnification images presented in the bottom rows. First column,co-staining with NCC (blue) in cortex showing distal convoluted tubulespositive for both, MUC1-fs and -wt. Second column, co-staining with AQP2(blue) in cortex and outer stripe of the outer medulla showingcollecting ducts positive for both, MUC1-fs and -wt. Third column,co-staining with LTL (blue) in cortex and outer stripe of the outermedulla showing lack of MUC1 staining with LTL-positive proximal tubulesin the cortex (8/9) and positive MUC1-fs staining in proximal 10 tubulesin the outer medulla (no MUC1-wt is detected). Fourth column,co-staining with AQP2 (blue) in inner medulla showing collecting ductspositive for both, MUC1-fs and -wt. FIG. 8B shows the characterizationof fs/+ mouse model, specifically presenting an IF image of a coronalsection from a 24 month old female fs/+ mouse, showing distributions ofMUC1-wt (red), MUC1-fs (green) and AQP2 (blue). Note severely dilatedtubules, particularly in the outer medulla were associated with highexpression of MUC1-fs (green).

FIGS. 9A to 9J depict the generation and characterization of MKD patientkidney organoids and immortalized tubular epithelial cell lines. FIG. 9Apresents IF images of human kidney organoids derived from iPSC cellsfrom a normal sibling or an affected MKD patient. Distribution ofMUC1-wt (red) and MUC1-fs (green) is shown in proximal and distaltubular structures, marked by LTL (yellow) and by E-cadherin (blue),respectively. FIG. 9B presents IF images of normal kidney-derivedepithelial cells (N) and MKD patient kidney-derived epithelial cells(P). Co-staining for MUC1-fs (green), MUC1-wt (red) and Hoechst (blue)showed that MUC1-fs is exclusively expressed in P cells and localizedintracellularly compared to MUC1-wt, which is expressed in both celllines and is localized to the plasma membrane. FIG. 9C shows immunoblotanalysis of MUC1-wt and MUC1-fs proteins in N and P cells, which showedMUC1-wt expression in both cell lines while MUC1-fs is expressed only inP cells. (n=5 replicates). FIG. 9D shows the results of RT-PCR analysisof P cells treated with THP (100 nM), which show that IRE inhibitor(IREi, 4μ8c) inhibited IRE activation, as detected by dose-dependentinhibition of XBP1 splicing (sXBP1). FIG. 9E shows immunoblot analysisof eIF2a in P cells, which showed that PERK inhibitor (PERM, GSK2656157)inhibited PERK activation, as detected by dose-dependent reduction ofeIF2a phosphorylation (p-eIF2a). FIG. 9F shows immunoblot analysis ofBiP in P cells treated with THP (100 nM), which showed that ATF6inhibitor (ATF6i, PF-429242) inhibited ATF6 activation, as detected bydose-dependent reduction of BiP abundance. FIG. 9G shows results fromthe quantitative live imaging analysis sequence of caspase 3/7activation. Following image acquisition (left panel), single cells werefirst identified using the digital phase contrast channel and cellnumber was calculated. Green fluorescence intensity was then measuredusing 488 channel (middle panel) and the threshold for Caspase3/7-positive staining was determined. As an output, the fraction ofcaspase 3/7-positive apoptotic cells (right panel, green) or caspase3/7-negative live cells (right panel, blue) was calculated in each wellat each time point. FIG. 9H shows immunoblot analysis of MUC1-fs in Pcells, which showed MUC1-fs accumulation following ATF6 inhibition(ATF6i, 10 μM) for 24 hours. (n=3 replicates). FIG. 9I is a set ofbright field images of representative N and P cells treated with DMSO orTHP (33 nM) overlaid with images of caspase 3/7 activation (apoptosis,green) show reduced cell number and increased proportion of caspasepositive P cells (quantification is shown in FIG. 9J). FIG. 9J showshigher susceptibility of P cells to THP-induced apoptosis after 72 hourtreatment with indicated concentrations of THP. Cell apoptosis wascalculated using the protocol summarized in FIG. 9G. Values are means+SD(n=4 replicates).

FIGS. 10A and 10B show that BRD-4780 was effective in removing mutantMUC1-fs from MKD patient kidney-derived epithelial cells. FIG. 10Apresents results from a primary screen. Primary screen statisticspresent the median and ±3 median absolute deviations for MUC1-fs IFquantification. Broad Repurposing Library 3713 compounds (grey), DMSOnegative control (orange) and JQ1 positive control (yellow). The Z′score statistic for this high content assay was 0.35. FIG. JOB showsresults from the RNA-Seq analysis of UPR branch activation (as in FIG.2A) in P cells pretreated with DMSO or BRD-4780 (1 μM) for 12 hours,followed by 12 hour co-treatment with THP (100 nM) (n=3 replicates).

FIGS. 11A to 11U show the pharmacokinetics (PK) of candidate compoundsobserved in 129S2 mice, 129S-ELITE mice, and Sprague Dawley rats.Compounds were delivered either intravenously (i.v.) or orally (p.o).FIG. 11A shows a plot of the plasma and kidney BRD-4780 concentrationvs. time curves in male 129S2 mice following a single i.v. or p.o. doseof BRD-4780 from a low dose PK study. FIG. 11B is a table of thecalculated PK parameters from the data plotted in FIG. 11A following asingle i.v. or p.o. dose of BRD-4780 in male 129S2 mice. “Rsq_adj”indicates the R-squared adjustment. “No.” indicates number. “C_(o)”indicates the initial extrapolated concentration. “C_(max)” indicatesthe maximum concentration. “T_(max)” indicates the time of maximumconcentration. “T_(1/2)” indicates the half-life. “V_(dss)” indicatesthe volume of distribution. “Cl” indicates clearance. “Inf” indicatesinfinity. Exrtrap” indicates extrapolated. “AUC” indicates the areaunder the curve. “AUC_(0-last) Ratio (K/P)” indicates the ratio ofAUC_(0-last) in kidney/AUC_(0-last) in plasma. FIG. 11C shows a plot ofthe plasma and kidney BRD-4780 concentration vs. time curves in malemice following a single p.o. dose of BRD-4780 at the indicated dosesfrom the dose response PK study. FIG. 11D shows a plot of the plasma andkidney BRD-4780 concentration vs. time curves in female mice following asingle p.o. dose of BRD-4780 at the indicated doses from the doseresponse PK study. FIG. 11E is a table of the calculated PK parametersfrom the data plotted in FIG. 11C following a single i.v. or p.o. doseof BRD-4780 at the indicated doses in male 129S2 mice. “Rsq_adj”indicates the R-squared adjustment. “No.” indicates number. “C_(o)”indicates the initial extrapolated concentration. “C_(max)” indicatesthe maximum concentration. “T_(max)” indicates the time of maximumconcentration. “T_(1/2)” indicates the half-life. “V_(dss)” indicatesthe volume of distribution. “Cl” indicates clearance. “Inf” indicatesinfinity. Exrtrap” indicates extrapolated. “AUC” indicates the areaunder the curve. “AUC_(0-last) Ratio (K/P)” indicates the ratio ofAUC_(0-last) in kidney/AUC_(0-last) in plasma. FIG. 11F is a table ofthe calculated PK parameters from the data plotted in FIG. 11D followinga single i.v. or p.o. dose of BRD-4780 at the indicated doses in female129S2 mice. “Rsq_adj” indicates the R-squared adjustment. “No.”indicates number. “C_(o)” indicates the initial extrapolatedconcentration. “C_(max)” indicates the maximum concentration. “T_(max)”indicates the time of maximum concentration. “T_(1/2)” indicates thehalf-life. “V_(dss)” indicates the volume of distribution. “Cl”indicates clearance. “Inf” indicates infinity. Exrtrap” indicatesextrapolated. “AUC” indicates the area under the curve. “AUC_(0-last)Ratio (K/P)” indicates the ratio of AUC_(0-last) in kidney/AUC_(0-last)in plasma. FIG. 11G shows a plot of the plasma BRD-4780 concentrationvs. time curves in male 129S-ELITE mice following a single i.v. or p.o.dose of BRD-4780 from a low dose PK study. FIG. 11H shows a plot of theplasma BRD-1365 (also referred to as “Fr1 (+)” herein) concentration vs.time curves in male 129S-ELITE mice following a single i.v. or p.o. doseof BRD-4780 from a low dose PK study. FIG. 11I shows a plot of theplasma BRD-7709 (also referred to as “Fr2 (−)” herein) concentration vs.time curves in male 129S-ELITE mice following a single i.v. or p.o. doseof BRD-4780 from a low dose PK study. FIG. 11J is a table of thecalculated PK parameters from the data plotted in FIGS. 11G to 11Ifollowing a single i.v. or p.o. dose of BRD-4780 at the indicated dosesin male 129S-ELITE mice. “C_(max)” indicates the maximum concentration.“T_(max)” indicates the time of maximum concentration. “AUC” indicatesthe area under the curve. “Inf” indicates infinity. “C_(max)D” indicatesthe dose normalized maximum concentration. T_(1/2) indicates thehalf-life. “Cl” indicates clearance. “C_(max)” indicates the maximumconcentration. “h” indicates hour. “Vz” indicates the volume ofdistribution. “AUC_(0-last) Ratio (K/P)” indicates the ratio ofAUC_(0-last) in kidney/AUC_(0-last) in plasma. FIG. 11K shows a plot ofthe plasma, kidney, liver, eye and brain BRD-4780 concentration vs. timecurves in male 129S-ELITE mice following a single p.o. dose of BRD-4780from a dose response PK study. FIG. 11L shows a plot of the plasma,kidney, liver, eye and brain BRD-1365 concentration vs. time curves inmale 129S-ELITE mice following a single p.o. dose of BRD-1365 from adose response PK study. FIG. 11M shows a plot of the plasma, kidney,liver, eye and brain BRD-7709 concentration vs. time curves in male129S-ELITE mice following a single p.o. dose of BRD-7709 from a doseresponse PK study. FIG. 11N is a table of the calculated PK parametersfrom the data plotted in FIG. 11K following a single p.o. dose ofBRD-4780 at the indicated doses in male 129S-ELITE mice. “C_(max)”indicates the maximum concentration. “T_(max)” indicates the time ofmaximum concentration. “AUC” indicates the area under the curve. “Inf”indicates infinity. “C_(max)D” indicates the dose normalized maximumconcentration. “T_(1/2)” indicates the half-life. “Cl” indicatesclearance. “C_(max)” indicates the maximum concentration. “h” indicateshour. “C_(max)” indicates the maximum concentration. “Vz” indicates thevolume of distribution. “AUC_(0-last) Ratio (K/P)” indicates the ratioof AUC_(0-last) in kidney/AUC_(0-last) in plasma FIG. 11O is a table ofthe calculated PK parameters from the data plotted in FIG. 11L followinga single p.o. dose of BRD-1365 at the indicated doses in male 129S-ELITEmice. “C_(max)” indicates the maximum concentration. “T_(max)” indicatesthe time of maximum concentration. “AUC” indicates the area under thecurve. “Inf” indicates infinity. “C_(max)D” indicates the dosenormalized maximum concentration. “T_(1/2)” indicates the half-life.“Cl” indicates clearance. “C_(max)” indicates the maximum concentration.“h” indicates hour. “C_(max)” indicates the maximum concentration. “Vz”indicates the volume of distribution. “AUC_(0-last) Ratio (K/P)”indicates the ratio of AUC_(0-last) in kidney/AUC_(0-last) in plasma.FIG. 11P is a table of the calculated PK parameters from the dataplotted in FIG. 11L following a single p.o. dose of BRD-7709 at theindicated doses in male 129S-ELITE mice. “C_(max)” indicates the maximumconcentration. “T_(max)” indicates the time of maximum concentration.“AUC” indicates the area under the curve. “Inf” indicates infinity.“C_(max)D” indicates the dose normalized maximum concentration.“T_(1/2)” indicates the half-life. “Cl” indicates clearance. C_(max)”indicates the maximum concentration. “h” indicates hour. “C_(max)”indicates the maximum concentration. “Vz” indicates the volume ofdistribution. “AUC_(0-last) Ratio (K/P)” indicates the ratio ofAUG_(0-last) in kidney/AUC_(0-last) in plasma. FIG. 11Q shows a plot ofthe plasma BRD-4780 concentration vs. time curves in male Sprague Dawleyrats following a single i.v. or p.o. dose of BRD-4780 from a low dose PKstudy. FIG. 11R shows a plot of the plasma BRD-1365 concentration vs.time curves in male Sprague Dawley rats following a single i.v. or p.o.dose of BRD-1365 from a low dose PK study. FIG. 11S shows a plot of theplasma BRD-7709 concentration vs. time curves in male Sprague Dawleyrats following a single i.v. or p.o. dose of BRD-7709 from a low dose PKstudy. FIG. 11T is a table of the calculated PK parameters from the dataplotted in FIGS. 11Q, 11R, and 11T following a single i.v. or p.o. doseof BRD-4780, BRD-1365 or BRD-7709 at the indicated doses in male SpragueDawley rats. “C_(max)” indicates the maximum concentration. “T_(max)”indicates the time of maximum concentration. “AUC” indicates the areaunder the curve. “Inf” indicates infinity. “C_(max)D” indicates the dosenormalized maximum concentration. “T_(1/2)” indicates the half-life.“C1” indicates clearance. “C_(max)” indicates the maximum concentration.“h” indicates hour. “C_(max)” indicates the maximum concentration. “Vz”indicates the volume of distribution. “AUC_(0-last) Ratio (K/P)”indicates the ratio of AUC_(0-last) in kidney/AUC_(0-last) in plasma.FIG. 11U shows a plot of the percent oral bioavailability of BRD-4780,BRD-1365 or BRD-7709 at the indicated doses in male Sprague Dawley ratsfrom a low dose PK study. FIG. 11V shows a plot of the mean and standarddeviation plasma BRD-4780 concentration vs. time curves in male CD(SD)rats following a single p.o. dose of BRD-4780 at 10 mg/kg p.o., 30 mg/kgp.o., 50 mg/kg p.o. or 100 mg/kg p.o. FIG. 11W shows a plot of the meanand standard deviation plasma BRD-4780 concentration vs. time curves infemale CD(SD) rats following a single p.o. dose of BRD-4780 at 10 mg/kgp.o., 30 mg/kg p.o., 50 mg/kg p.o. or 100 mg/kg p.o. FIG. 11X shows aplot of the mean and standard deviation plasma BRD-7709 concentrationvs. time curves in male CD(SD) rats following a single p.o. dose ofBRD-7709 at 10 mg/kg p.o., 30 mg/kg p.o., 50 mg/kg p.o. or 100 mg/kgp.o. FIG. 11Y shows a plot of the mean and standard deviation plasmaBRD-7709 concentration vs. time curves in female CD(SD) rats following asingle p.o. dose of BRD-7709 at 10 mg/kg p.o., 30 mg/kg p.o., 50 mg/kgp.o. or 100 mg/kg p.o. FIG. 11Z shows a plot of the mean and standarddeviation plasma BRD-1365 concentration vs. time curves in male CD(SD)rats following a single p.o. dose of BRD-1365 at 10 mg/kg p.o., 30 mg/kgp.o., 50 mg/kg p.o. or 100 mg/kg p.o. FIG. 11AA shows a plot of the meanand standard deviation plasma BRD-1365 concentration vs. time curves infemale CD(SD) rats following a single p.o. dose of BRD-1365 at 10 mg/kgp.o., 30 mg/kg p.o., 50 mg/kg p.o. or 100 mg/kg p.o. FIG. 11AB is atable of calculated PK parameters from the data plotted in FIG. 11V andFIG. 11W following a single p.o. dose of BRD-4780 at the indicated dosesin male and female CD(SD) rats. “C_(max)” indicates the maximumconcentration. “T_(max)” indicates the time of maximum concentration.“C_(last)” indicates the last measured concentration. “T_(last)”indicates the time of last detected plasma concentration. “T_(1/2)”indicates the half-life. “AUC₀₋₂₄” indicates the area under curve up to24 hours. “AUC_(last)” indicates the area under the curve up to lastmeasured time point. “AUC_(0-inf)” indicates the extrapolated area underthe curve. “AUC_(%) Extrap” indicates the percentage of the area of thecurve that is extrapolated. “CD” indicates the dose normalized C_(max).“AUC_(0-24/D)” indicates the dose normalized area of the curve to 24hours. “AUC_(last/D)” indicates the dose normalized area of the curve tolast time point. “CL” indicates clearance. “V_(z)” indicates the volumeof distribution. FIG. 11AC is a table of calculated PK parameters fromthe data plotted in FIG. 11X and FIG. 11Y following a single p.o. doseof BRD-7709 at the indicated doses in male and female CD(SD) rats.“C_(max)” indicates the maximum concentration. “T_(max)” indicates thetime of maximum concentration. “C_(last)” indicates the last measuredconcentration. “T_(last)” indicates the time of last detected plasmaconcentration. “T_(1/2)” indicates the half-life. “AUC₀₋₂₄” indicatesthe area under curve up to 24 hours. “AUC_(last)” indicates the areaunder the curve up to last measured time point. “AUC_(0-inf)” indicatesthe extrapolated area under the curve. “AUC_(%) Extrap” indicates thepercentage of the area of the curve that is extrapolated. “C_(D)”indicates the dose normalized Cmax. “AUC_(0-24/D)” indicates the dosenormalized area of the curve to 24 hours. “AUC_(last/D)” indicates thedose normalized area of the curve to last time point. “CL” indicatesclearance. “V_(z)” indicates the volume of distribution. FIG. 11AD is atable of calculated PK parameters from the data plotted in FIG. 11Z andFIG. 11AA following a single p.o. dose of BRD-1365 at the indicateddoses in male and female CD(SD) rats. “C_(max)” indicates the maximumconcentration. “T_(max).” indicates the time of maximum concentration.“C_(last)” indicates last measured concentration. “T_(last)” indicatesthe time of last detected plasma concentration, where no data (ND) islisted if T_(last) is not equal for the 3 rats. “T_(1/2)” indicates thehalf-life. “AUC₀₋₂₄” indicates the area under the curve for up to 24hours. “AUC_(0-inf)” indicates the extrapolated area under the curve.“AUC_(%) Extrap” indicates the percentage of the area of the curve thatis extrapolated. “C_(D)” indicates the dose normalized Cmax.“AUC_(0-24/D)” indicates the dose normalized area of the curve up to 24hours. “AUC_(last/D)” indicates the dose normalized area of the curve tolast time point. “CL” indicates clearance. “V_(z)” indicates volume ofdistribution.

FIGS. 12A and 12B show MUC1 immunofluoresence (IF) staining in kidneysections from fs/+ mice and in iPSC-derived kidney organoids. FIG. 12Adepicts mean MUC1-wt IF intensity in NCC-positive cells in kidneysections from fs/+ mice treated with vehicle or BRD-4780 (1, 10 and 50mg/kg) for 7 days. Values are means+SD (see Example 1 below for details;n=4 mice/genotype/dose). FIG. 12B shows results of the quantitativeregion-of-interest IF analysis sequence performed in FIGS. 4F and 4G.Image acquisition of an entire organoid section (original image of asingle field), was performed for MUC1-fs (green), MUC1-wt (red) andlaminin (blue) staining. MUC1-wt positive regions were identified usingMUC1-wt staining (red) using a threshold of >7000 intensity and >2500μm². MUC1-wt mean intensity was subsequently calculated in these regionsand averaged for the entire organoid section. MUC1-wt regions, whichwere <5 μm apart, were clustered together and an area of 20 μmsurrounding these clusters was selected as a MUC1 positive tubule.MUC1-fs signal was then detected using a spot identifier within thesetubules excluding laminin area; the mean intensity of FS signal wascalculated there and averaged for the entire organoid section.

FIGS. 13A to 13D show that MUC1-fs accumulates in the early secretorypathway, in a TMED9 cargo receptor-positive compartment. FIG. 13A showsresults of the IF analysis sequence of MUC1-fs intracellularco-localization in P cells. Following image acquisition of MUC1-fs(green) and organelle (red), single cell cytoplasm was identified (whiteborder lines)(bottom right and middle panels) and the signals of MUC1-fsand the organelle within the cytoplasm was detected and displayed asspots of different colors (bottom left and middle, respectively).Overlap between MUC1-fs signal (bottom right, green) and the organellesignal (bottom right, red) was obtained only when pixels of the twosignals (green and red) overlapped (bottom left, yellow).Co-localization was then calculated as total overlap area, normalized(divided) to the total area of MUC1-fs, and then to the total area ofthe organelle. FIG. 13B presents representative IF images of MUC1-fscolocalization with organelle-specific markers (as calculated in FIG.5B). Right panel shows the software reconstruction that was used fordownstream measurements (MUC1-fs, green; organelle, red; overlap,yellow). FIG. 13C depicts the effects of BFA and of Bafilomycin A onMUC1-fs subcellular distribution. P cells were treated with BFA (200ng/mL) or Bafilomycin A (100 nM) for 24 hours and MUC1-fs distributionwas tested as described in FIG. 5B. Percent change of DMSO treated cellswas calculated for MUC1-fs co-localization with each organelle marker(n=3 replicates). Treatment with BFA resulted in MUC1-fs accumulation inthe ER (and reduction in the late secretory pathway i.e. cis- andtrans-Golgi, and endosomes). Treatment with Bafilomycin A resulted inaccumulation of MUC1-fs in the late secretory pathway, and especiallythe late endosome/lysosome). FIG. 13D shows immunoblot analysis ofMUC1-fs in P cells following 5 hour inhibition of the proteosome byBortezomib (50 nM) in the absence or presence of BRD-4780 (5 μM) (left).Proteasomal inhibition was confirmed in N cells by increased ubiquitinlevels after Bortezomib treatment (50 nM) (right). Proteosomalinhibition did not affect MUC1-fs removal by BRD-4780, indicating thatits degradation did not occur in the proteasome (n=3 replicates).

FIGS. 14A to 14E depict that BRD-4780 was identified to act byengagement of its target, the cargo receptor TMED9. FIG. 14A presents IFimages of MUC1-fs (green), GM130 (red) and MUC1-wt (blue) in MKD patientiPSC-derived kidney organoids, which showed no change in GM130 abundanceupon BRD-4780 (10 μM) treatment for 72 hour. FIG. 14B presents IF imagesof MUC1-wt (yellow) and Hoechst (grey) in P cells after TMED9 orNischarin deletion, as compared to cells treated with non-targetingsgRNA control (NTC) before and after treatment with BRD-4780 (5 μM) for72 hours. No change in either MUC1-wt abundance or its plasma membranelocalization was observed. FIG. 14C shows immunoblot analysis of P cellsafter depletion of I1R candidate (Nischarin) using shRNAs (KD1 and KD2;top) or CRISPR-Cas9 deletion (KO1 and KO2; bottom). BRD-4780 (5 μM)treatment was applied for 72 hours. BRD-4780 remained effective despiteI1R depletion. FIG. 14D shows a table of eighteen compounds annotated asimidazoline-1 receptor ligands (IRLs) and their EC₅₀ values forreduction in MUC1-fs levels. Each compound was applied to P cells for 48hours and effects on MUC1-fs were analyzed by IF imaging. (N/A,non-active, EC50>2E-05 [M]). FIG. 14E shows that BRD-4780 did not bindNischarin, as assessed by CETSA. Densitometric analysis of Nischarinabundance after treatment of P cells with BRD-4780 (5 μM, 1 hour) orDMSO, followed by exposure to escalating temperatures showed no changein nischarin abundance in the presence of BRD-4780. Solid linesrepresent the best fits of the data to the Boltzmann sigmoid. Values aremeans±SEM. (n=3 replicates).

FIGS. 15A to 15H show that BRD-4780 was effective in removing severalmisfolded proteins. FIG. 15A shows IF images of UMOD (green) and Hoechst(grey) in AtT-20 cells stably transfected with C126R-UMOD and treatedfor 72 hours with DMSO or BRD-4780 (10 μM). FIG. 15B presents results ofIF quantification of C126R-UMOD in AtT-20 cells treated as in FIG. 4A.Values are means±SD (n=4 replicates). FIG. 15C shows an immunoblotanalysis of UMOD and ERp72 in AtT-20 cells stably transfected withC126R-UMOD, pre-treated for 24 hours with DMSO or BRD-4780 (1 μM or 10μM) and an additional 24 hours with THP (10 nM). FIG. 15D presentsbright field (BF) images (grey), overlaid with images of mutantrhodopsin P23H-GFP (green) in N cells pre-treated for 48 hours with DMSOor BRD-4780 (5 μM) followed by transient transfection with mutantrhodopsin P23H-GFP. FIG. 15E presents results of GFP intensity in live Ncells expressing P23H-GFP and treated as in FIG. 15D (see Example 1below for details). Values are means±SD. (n=8 replicates). FIG. 15Fshows the results of an experiment in which the fraction ofDRAQ7-positive cells (cell death marker) was determined in live N cellstransiently transfected with mutant rhodopsin P23H-GFP and treated withDMSO or BRD-4780 (5 μM) for 72 hours. Values are means±SD. (n=8replicates). FIG. 15G shows bright field (BF) images (grey), overlaidwith images of huntingtin-GFP containing 97 polyQ repeats (green) in HEKcells transiently transfected with huntingtin-GFP containing 97 polyQrepeats and treated for 72 hours with DMSO or BRD-4780 (10 μM). FIG. 15Hpresents results of GFP puncta fluorescence intensity of huntingtinprotein in live HEK cells expressing huntingtin-GFP and treated as inFIG. 15G (see Example 1 below for details). Values are means±SD (n=3replicates).

FIG. 16 presents a table which shows how BRD-4780 has drug-likeproperties, as demonstrated by target profile criteria. LE, Ligandefficiency. LLE, Ligand lipophilic efficiency. PO, per os.

FIG. 17 shows a listing of serum creatinine levels in +/+ and fs/+ miceas a function of age and gender (see FIG. 7E above).

FIG. 18 shows structures of amino functionalized BRD-4780 compounds(identified as compounds 3-11 successively, where all such compounds areracemates), as described in Example 15 below.

FIG. 19 shows a chromatogram that demonstrates the best separation ofp-NO₂-Cbz 5 with stationary phase AD-H and mobile phase iPrOH (3-50%).

FIGS. 20A and 20B show that ¹H-NMR spectra of enantiopure Mosher amidespresented distinct display diagnostic chemical shift differences. FIG.20A shows fraction 1 (Fr1) results. FIG. 20B shows fraction 2 (Fr2)results.

FIGS. 21A and 21B show rhodopsin distribution in mouse retinal sectionsin response to BRD-4780. FIG. 21A shows representative images of retinalsections from mice (Rho/+ or +/+) treated with vehicle or 50 mg/Kg/dayBRD-4780 for 28 days. Rhodopsin antibody staining is shown in green andDAPI staining of nuclei is shown in blue. The arrows indicate the outersegment (OS) of the photoreceptors; the asterisks indicate the outernuclear layer (ONL) of the photoreceptors. Bars=100 μm. FIG. 21B showsquantification of rhodopsin staining in the ONL normalized to the numberof nuclei. Rhodopsin mainly localizes to the OS of the photoreceptors,therefore changes observed in the staining in the ONL bona fidereflected changes in the accumulation of rhodopsin in intracellularcompartments.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is directed, at least in part, to the discoverythat a small molecule, 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane(also referred to as BRD-4780 herein and previously known as AGN192403), was capable of reversing and therefore treating a number oftoxic proteinopathies, via a mechanism involving the clearance of mutantproteins (that would otherwise provoke proteinopathies via extendedresidence within an affected cell, tissue, organoid and/or subject).

Compositions and methods for treatment of toxic proteinopathies (e.g.,MKD (e.g., due to a frameshift or other mutation in MUC1), RetinitisPigmentosa (e.g., due to rhodopsin mutations), autosomal dominanttubulo-interstitial kidney disease due to UMOD mutations (ADTKD-UMOD),and other forms of toxic proteinopathies resulting from mutant proteinaccumulation in the ER and/or other secretory pathway compartmentsand/or vesicles) are described in additional detail below.

Intracellular accumulation of misfolded proteins causes toxicproteinopathies, diseases which have been heretofore lacking intreatment options. MUC1 kidney disease (MKD) results from a frameshiftmutation in the mucin 1 gene (MUC1-fs). The instant disclosure hasidentified that MKD is a toxic proteinopathy. As has been demonstratedherein, intracellular MUC1-fs accumulation activated the ATF6 unfoldedprotein response (UPR) branch. A small molecule, BRD-4780, was thenidentified via screening to be capable of clearing MUC1-fs from patientcells, from kidneys of knock-in mice and from patient kidney organoids.MUC1-fs was trapped in TMED9 cargo receptor-containing vesicles of theearly secretory pathway. Without wishing to be bound by theory, BRD-4780was identified herein to bind TMED9, releasing MUC1-fs and re-routing itfor lysosomal degradation, an effect that was found to be phenocopied byTMED9 deletion. The instant disclosure has therefore identified BRD-4780as a promising therapeutic lead compound for treatment of MKD. BRD-4780also diminished mutant proteins associated with two additional toxicproteinopathies in in vitro studies. In certain aspects, the instantdisclosure has therefore generally elucidated a novel therapeuticstrategy for the release of misfolded, truncated or mutated proteinsfrom cargo receptors and their anterograde trafficking to the lysosome.

Toxic Proteinopathies and Protein Trafficking

Diseases associated with protein misfolding and aggregation are known asproteinopathies (Bayer, 2015). More than 50 proteinopathies are causedby genetic mutations that result in protein misfolding and intracellularaccumulation (Dubnikov et al., 2017; Dugger and Dickson, 2017). Theaccumulated proteins can cause cellular toxicity, as seen in some formsof Amyotrophic Lateral Sclerosis (ALS), Parkinson's disease, andRetinitis Pigmentosa (RP) (Dubnikov et al., 2017; Dugger and Dickson,2017). Some proteinopathies, such as RP (Athanasiou et al., 2018), arecharacterized by the accumulation of misfolded proteins in the earlysecretory pathway (endoplasmic reticulum (ER) and Golgi apparatus),whereas others, such as Huntington's disease (Zoghbi and Orr, 2000),involve accumulation of misfolded protein aggregates in the cytoplasm orthe nucleus.

The early secretory pathway of eukaryotic cells is composed of threeorganelles, the ER, the ER-Golgi intermediate compartment (ERGIC; alsoincluding COPI- and COPII coated transport vesicles), and the Golgiapparatus (Gomez-Navarro and Miller, 2016; including the cis-Golgi,medial cisternae of the Golgi apparatus, and the trans-Golgi network(TGN), also including Golgi transport vesicles). The ER is amultifunctional organelle that orchestrates the synthesis, folding, andstructural maturation of nearly one third of all cellular proteins (Hetzet al., 2015). The Golgi apparatus occupies a central position withinthe secretory pathway, acting as a hub for vesicular trafficking.Distinct classes of vesicles transport diverse cargoes into and out ofthis organelle, as well as between the cis- and trans-Golgisub-compartments (Gomez-Navarro and Miller, 2016; Witkos and Lowe,2017). Maintenance of the ER and Golgi apparatus requires a balance ofanterograde coat protein II (COPII)-mediated and retrograde(COPI)-mediated vesicular trafficking (Gomez-Navarro and Miller, 2016).Together, the ER and the Golgi are responsible for biogenesis and properintracellular distribution of a wide range of proteins (Gomez-Navarroand Miller, 2016).

The Unfolded Protein Response (UPR) is activated upon increasedsecretory protein load to ensure maintenance of cellular homeostasis(Brandizzi and Barlowe, 2013; Plate and Wiseman, 2017; Walter and Ron,2011). Mutant proteins disrupt the secretory pathway and trigger the UPR(Walter and Ron, 2011). The three principal branches of the UPR, IRE1(inositol requiring enzyme), PERK (PKR-like ER kinase), and ATF6(activating transcription factor 6) work together to maintain ERhomeostasis (Walter and Ron, 2011). However, in the setting of excess orprolonged cellular stress, the protective capacity of the UPR may beinsufficient to restore homeostasis, triggering the induction of celldeath (Walter and Ron, 2011), a hallmark of many proteinopathies(Remondelli and Renna, 2017).

Autosomal Dominant Tubulo-interstitial Kidney Disease-Mucin1 (ADTKD-MUC1or MUC1 kidney disease, MKD) is caused by a frameshift in the GC-richVariable Number of Tandem Repeats (VNTR) region of the MUC1 gene (Kirbyet al., 2013). MKD is characterized by slowly progressivetubulo-interstitial disease that leads to kidney failure (Bleyer et al.,2017; Yu et al., 2018). Affected individuals develop kidney failure bythe second to seventh decade of life, requiring kidney replacementprocedures (dialysis) complicated by high mortality rates, or kidneytransplantation complicated by chronic immunosuppression and associatedtoxicities (Bleyer et al., 2017; Yu et al., 2018).

The MUC1 gene encodes the transmembrane glycoprotein mucin 1 (MUC1),which is expressed at the apical surface of glandular or luminalepithelial cells in the mammary gland, digestive tract, uterus,prostate, lung and kidney (Hattrup and Gendler, 2008). MUC1 has beenmainly studied in epithelial cancers (Nath and Mukherjee, 2014). In thehealthy adult kidney, MUC1 localizes to distal convoluted tubule andcollecting duct (Leroy et al., 2002), while following ischemia, theprotein may be induced in the proximal tubule (Al-bataineh et al., 2016;Gibier et al., 2017). As a plasma membrane protein, MUC1 is synthesizedand core-glycosylated in the ER, followed by extensive O-glycosylationof its VNTR region in the Golgi apparatus (Hilkens and Buijs, 1988;Litvinov and Hilkens, 1993).

In all known cases of MKD, the causative mutations result in the sameframeshift, producing a mutant MUC1 neo-protein (MUC1-fs) (Kirby et al.,2013; Wenzel et al., 2018; Yamamoto et al., 2017; Živná et al., 2018).The vast majority of these mutations involve the insertion of an extracytosine in a string of seven cytosines within one of the VNTR subunits(Kirby et al., 2013). MUC1-fs retains the wild-type N-terminal signalsequence that drives ER translation, but beyond the insertion, it hastandem series of novel 20 amino acid imperfect repeats and a C-terminalneo-peptide with an early stop codon, resulting in the absence of thetransmembrane and intracellular domains found in the wild-type MUC1protein (Kirby et al., 2013; FIGS. 7A and 7B). The molecular mechanismresponsible for MKD has been heretofore unknown and no therapy has beenpreviously available for MKD.

The instant disclosure has established that intracellular accumulationof MUC1-fs in early secretory compartments leads to activation of theUPR. Also described herein is the identification of a small molecule,BRD-4780 (2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane), thatclears mutant MUC1-fs in patient cells, knock-in mouse kidneys andpatient iPSC-derived kidney organoids. BRD-4780(2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane), has additionallybeen described herein to release mutant MUC1-fs trapped in TMED9 cargoreceptor-enriched and cis-Golgi compartments of the early secretorypathway in patient cells, knock-in mouse kidneys and patientiPSC-derived kidney organoids. Without wishing to be bound by theory,BRD-4780 thus promotes MUC1-fs anterograde trafficking toward lysosomaldegradation. TMED9 deletion was discovered to phenocopy the effect ofBRD-4780. The instant disclosure has therefore elucidated the cellularmechanism underlying MKD and has identified BRD-4780 as a promising leadfor the treatment of toxic proteinopathies.

MUC1-Associated Kidney Disease (MKD, aka Autosomal DominantTubulointerstitial Kidney Disease, MUC1-Related or ADTKD-MUC1)

MUC1-Associated Kidney Disease (MKD), also known as autosomal dominanttubulointerstitial kidney disease, MUC1-related (ADTKD-MUC1), waspreviously known as medullary cystic kidney disease type 1. It ischaracterized by slowly progressive tubulointerstitial disease thatleads to end-stage renal disease (ESRD) and the need for dialysis orkidney transplantation. ESRD typically occurs in adulthood but isextremely variable, occurring at any age between 20 and 70 years. Thereare no other systemic manifestations (Bleyer and Kmoch. Gene Reviews2016).

Autosomal Dominant Tubulointerstitial Kidney Disease Caused by UMODPathogenic Variants (ADTKD-UMOD)

Autosomal dominant tubulointerstitial kidney disease caused by UMODpathogenic variants (ADTKD-UMOD) was previously known as familialjuvenile hyperuricemic nephropathy type 1 (FJHN1), medullary cystickidney disease type 2 (MCKD2), and UMOD-associated kidney disease (oruromodulin-associated kidney disease). Typical clinical findings includeurinalysis revealing minimal protein and no blood; slowly progressivechronic kidney failure, usually first noted in the teen years andprogressing to end-stage renal disease (ESRD) between the fourth andseventh decades (Age at ESRD varies among and within families.); andhyperuricemia and gout (resulting from reduced kidney excretion of uricacid) that occurs as early as the teenage years (Bleyer et al. GeneReviews).

Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a genetic disorder of the eyes that causesloss of vision (“Facts About Retinitis Pigmentosa”. National EyeInstitute. May 2014). Symptoms include trouble seeing at night anddecreased peripheral vision (side vision; “Facts About RetinitisPigmentosa”. National Eye Institute. May 2014). Onset of symptoms isgenerally gradual (Understanding Retinitis Pigmentosa (PDF). Universityof Michigan Kellogg Eye Center). As peripheral vision worsens, peoplemay experience “tunnel vision”. Complete blindness is uncommon(Understanding Retinitis Pigmentosa).

Retinitis pigmentosa is generally inherited from a person's parents.Mutations in one of more than 50 genes is involved. The underlyingmechanism involves the progressive loss of rod photoreceptor cells inthe back of the eye. This is generally followed by loss of conephotoreceptor cells. Diagnosis is by an examination of the retinafinding dark pigment deposits. Other supportive testing may include anelectroretinogram, visual field testing, or genetic testing (“FactsAbout Retinitis Pigmentosa”).

There is currently no cure for retinitis pigmentosa (UnderstandingRetinitis Pigmentosa). Efforts to manage the problem may include the useof low vision aids, portable lighting, or a guide dog. Vitamin Apalmitate supplements may be useful to slow worsening. A visualprosthesis may be an option in certain people with severe disease. It isestimated to affect 1 in 4,000 people. Onset is often in childhood butsome are not affected until adulthood (“Facts About RetinitisPigmentosa”; Understanding Retinitis Pigmentosa).

The initial retinal degenerative symptoms of retinitis pigmentosa arecharacterized by decreased night vision (nyctalopia) and the loss of themid-peripheral visual field (Shintani et al. Optometry. 80: 384-401).The rod photoreceptor cells, which are responsible for low-light visionand are orientated in the retinal periphery, are the retinal processesaffected first during non-syndromic forms of this disease (Soucy et al.Neuron. 21: 481-93). Visual decline progresses relatively quickly to thefar peripheral field, eventually extending into the central visual fieldas tunnel vision increases. Visual acuity and color vision can becomecompromised due to accompanying abnormalities in the cone photoreceptorcells, which are responsible for color vision, visual acuity, and sightin the central visual field (Soucy et al. Neuron. 21: 481-93). Theprogression of disease symptoms occurs in a symmetrical manner, withboth the left and right eyes experiencing symptoms at a similar rate(Hartong et al. The Lancet. 368: 1795-1809).

A variety of indirect symptoms characterize retinitis pigmentosa alongwith the direct effects of the initial rod photoreceptor degenerationand later cone photoreceptor decline. Phenomena such as photophobia,which describes the event in which light is perceived as an intenseglare, and photopsia, the presence of blinking or shimmering lightswithin the visual field, often manifest during the later stages of RP.Findings related to RP have often been characterized in the fundus ofthe eye as the “ophthalamic triad”. This includes the development of (1)a mottled appearance of the retinal pigment epithelium (RPE) caused bybone spicule formation, (2) a waxy appearance of the optic nerve, and(3) the attentuation of blood vessels in the retina (Shintani et al.Optometry. 80: 384-401).

Non-syndromic RP usually presents a variety of the following symptoms:night blindness; tunnel vision (due to loss of peripheral vision);latticework vision; photopsia (blinking/shimmering lights); photophobia(aversion to bright lights); development of bone spicules in the fundus;slow adjustment from dark to light environments and vice versa; blurringof vision; poor color separation; loss of central vision; and eventualblindness.

A variety of retinal molecular pathway defects have been matched tomultiple known RP gene mutations. Mutations in the rhodopsin gene, whichis responsible for the majority of autosomal-dominantly inherited RPcases, disrupts the rod-opsin protein essential for translating lightinto decipherable electrical signals within the phototransductioncascade of the central nervous system. Defects in the activity of thisG-protein-coupled receptor are classified into distinct classes thatdepend on the specific folding abnormality and the resulting molecularpathway defects. The Class I mutant protein's activity is compromised asspecific point mutations in the protein-coding amino acid sequenceaffect the pigment protein's transportation into the outer segment ofthe eye, where the phototransduction cascade is localized. Additionally,the misfolding of Class II rhodopsin gene mutations disrupts theprotein's conjunction with 11-cis-retinal to induce proper chromophoreformation. Additional mutants in this pigment-encoding gene affectprotein stability, disrupt mRNA integrity post-translationally, andaffect the activation rates of transducin and opsin optical proteins(Mendes et al. Trends in Molecular Medicine. 11: 177-185).

Additionally, animal models suggest that the retinal pigment epitheliumfails to phagocytose the outer rod segment discs that have been shed,leading to an accumulation of outer rod segment debris. In mice that arehomozygous recessive for retinal degeneration mutation, rodphotoreceptors stop developing and undergo degeneration before cellularmaturation completes. A defect in cGMP-phosphodiesterase has also beendocumented; this leads to toxic levels of cGMP.

An accurate diagnosis of retinitis pigmentosa relies on thedocumentation of the progressive loss photoreceptor cell function,confirmed by a combination of visual field and visual acuity tests,fundus and optical coherence imagery, and electroretinography (ERG).

Visual field and acuity tests measure and compare the size of thepatient's field of vision and the clarity of their visual perceptionwith the standard visual measurements associated with healthy 20/20vision. Clinical diagnostic features indicative of retinitis pigmentosainclude a substantially small and progressively decreasing visual areain the visual field test, and compromised levels of clarity measuredduring the visual acuity test (Abigail T Fahim. “Retinitis PigmentosaOverview”). Additionally, optical tomography such as fundus and retinal(optical coherence) imagery provide further diagnostic tools whendetermining an RP diagnosis. Photographing the back of the dilated eyeallows the confirmation of bone spicule accumulation in the fundus,which presents during the later stages of RP retinal degeneration.Combined with cross-sectional imagery of optical coherence tomography,which provides clues into photoreceptor thickness, retinal layermorphology, and retinal pigment epithelium physiology, fundus imagerycan help determine the state of RP progression (Chang et al. CurrentGenomics. 12: 267-75).

While visual field and acuity test results combined with retinal imagerysupport the diagnosis of retinitis pigmentosa, additional testing isnecessary to confirm other pathological features of this disease.Electroretinography (ERG) confirms the RP diagnosis by evaluatingfunctional aspects associated with photoreceptor degeneration, and candetect physiological abnormalities before the initial manifestation ofsymptoms. An electrode lens is applied to the eye as photoreceptorresponse to varying degrees of quick light pulses is measured. Patientsexhibiting the retinitis pigmentosa phenotype would show decreased ordelayed electrical response in the rod photoreceptors, as well aspossibly compromised cone photoreceptor cell response(cdn.intechopen.com/pdfs-wm/17267.pdf).

There is currently no cure for retinitis pigmentosa, but the efficacyand safety of various prospective treatments are currently beingevaluated.

It is explicitly contemplated that toxic proteinopathies amenable totreatment with a TMED9-binding agent, e.g., BRD-4780, include, withoutlimitation, MUC1 kidney disease (Autosomal Dominant TubulointerstitialKidney Disease—MUC1, with frameshift MUC1 as the major aggregatingprotein), uromodulin kidney disease (Autosomal DominantTubulointerstitial Kidney Disease—UMOD, with mutant uromodulin as themajor aggregating protein), retinitis pigmentosa due to rhodopsinmutations (Rhodopsin mutations as the major aggregating protein),Parkinson's disease and other synucleinopathies—mutations inalpha-synuclein (α-Synuclein mutations as the major aggregatingprotein), familial Danish dementia due to ADan amyloid protein, CADASIL(cerebral autosomal dominant arteriopathy with subcortical infarcts andleukoencephalopathy) due to Notch3 mutations, seipinopathies due toseipin, serpinopathies (multiple) due to serpin, type II diabetes due toislet amylin polypeptide, lysozyme amyloidosis due to lysozyme, dialysisamyloidosis due to beta2 microglobumin, cataracts due to crystallins,odontogenic tumor amyloid due to Odontogenic ameloblast-associatedprotein, familial British dementia due to ABri amyloid protein,hereditary cerebral hemorrhage with amyloidosis (Icelandic) due toCystatin C, familial amyloidotic neuropathy or Senile systemicamyloidosis due to Transthyretin, ApoAII amyloidosis due to ApoAIIprotein, familial amyloidosis of the Finnish type (FAF) due to Gelsolin,fibrinogen amyloidosis due to Fibrinogen, inclusion bodymyositis/myopathy due to Amyloid β peptide (Aβ), hereditary latticecorneal dystrophy due to Keratoepithelin, pulmonary alveolar proteinosisdue to Surfactant protein C (SP-C), cystic fibrosis due to CFTRmutations, and any other disease or disorder in which a misfoldedprotein is trapped in the ER, COPI, COPII, ERGIC and/or Golgicompartments and/or early secretory pathway transport vesicles.

Identification of Proteinopathy in a Cell, Tissue and/or Subject

Identification of a cell and/or tissue of a subject as exhibiting aproteinopathy can be performed by any method available in the art. Suchmethods include those for assessing kidney, eye, nervous system, etc.function and/or for diagnosing kidney disease (e.g., MKD, ADTKD-UMOD,etc.) and/or RP. Evaluation of a subject for kidney function caninclude, for example, urinalysis (e.g., assessment of protein or bloodin the urine), any signs of kidney failure, etc. Evaluation of a subjectfor eye function that might be indicative of RP can be performed viaart-recognized methods of assessing for RP (e.g., diagnosis by anexamination of the retina finding dark pigment deposits, assessment byelectroretinogram, visual field testing, or genetic testing) and/or oneor more symptoms of RP (e.g., night blindness; tunnel vision (due toloss of peripheral vision); latticework vision; photopsia(blinking/shimmering lights); photophobia (aversion to bright lights);development of bone spicules in the fundus; slow adjustment from dark tolight environments and vice versa; blurring of vision; poor colorseparation; loss of central vision; and/or eventual blindness).

In certain embodiments, detection of a mutant locus and/or encodedprotein is performed, including, e.g., detection of one or more of thefollowing: a MUC1 frameshift mutant, a UMOD mutant characterized by ERretention (e.g., C126R UMOD) and/or a rhodopsin mutant characterized byER retention (e.g., P23H rhodopsin) and/or other gene or gene productassociated with a toxic proteinopathy.

Exemplary proteinopathy-associated mutations of the instant disclosureinclude the above-referenced MUC1 frameshift mutation, C126R UMODmutation and P23H rhodopsin mutation. Sequences for these mutations areprovided immediately below.

cDNA sequence of MUC1-fs (mutation site in lower case) (SEQ ID NO: 1)ATGACACCGGGCACCCAGTCTCCTTTCTTCCTGCTGCTGCTCCTCACAGTGCTTACAGTTGTTACgGGTTCTGGTCATGCAAGCTCTACCCCAGGTGGAGAAAAGGAGACTTCGGCTACCCAGAGAAGTTCAGTGCCCAGCTCTACTGAGAAGAATGCTGTGAGTATGACCAGCAGCGTACTCTCCAGCCACAGCCCCGGTTCAGGCTCCTCCACCACTCAGGGACAGGATGTCACTCTGGCCCCGGCCACGGAACCAGCTTCAGGTTCAGCTGCCACCTGGGGACAGGATGTCACCTCGGTCCCAGTCACCAGGCCAGCCCTGGGCTCCACCACCCCACCAGCCCACGATGTCACCTCAGCCCCGGACAACAAGCCAGCCCCGGGCTCCACCGCCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCAAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCCGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGAGAGCAGGCCGGCCCCGGGCTCCACCGCGCCCGCAGCCCACGGTGTCACCTCGGCCCCGGAGAGCAGGCCGGCCCCGGGCTCCACCGCGCCCGCAGCCCACGGTGTCACCTCGGCCCCGGAGAGCAGGCCGGCCCCGGGCTCCACCGCGCCCGCAGCCCACGGTGTCACCTCGGCCCCGGAGAGCAGGCCGGCCCCGGGCTCCACCGCGCCCGCAGCCCACGGTGTCACCTCGGCCCCGGAGAGCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGAGAGCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGAGAGCAGGCCGGCCCCGGGCTCCACCGCGCCCGCAGCCCACGGTGTCACCTCGGCCCCGGAGAGCAGGCCGGCCCCGGGCTCCACCGCGCCCGCAGCCCACGGTGTCACCTCGGCCCCGGAGAGCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCGCCCGCAGCCCACGGTGTCACCTCGGCCCCGGAGAGCAGGCCGGCCCCGGGCTCCACCGCGCCCGCAGCCCACGGTGTCACCTCGGCCCCGGAGAGCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCGGGCCCCGGGCTCCACCCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTTGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCATGGTGTCACCTCGGCCCCGGACAACAGGCCCGCCTTGGGCTCCACCGCCCCTCCAGTCCACAATGTCACCTCGGCCTCAGGCTCTGCATCAGGCTCAGCTTCTACTCTGGTGCACAACGGCACCTCTGCCAGGGCTACCACAACCCCAGCCAGCAAGAGCACTCCATTCTCAATTCCCAGCCACCACTCTGATACTCCTACCACCCTTGCCAGCCATAGCACCAAGACTGATGCCAGTAGCACTCACCATAGCTCGGTACCTCCTCTCACCTCCTCCAATCACAGCACTTCTCCCCAGTTGTCTACTGGGGTCTCTTTCTTTTTCCTGTCTTTTCACATTTCAAACCTCCAGTTTAATTCCTCTCTGGAAGATCCCAGCACCGACTACTACCAAGAGCTGCAGAGAGACATTTCTGAAATGTTTTTGCAGATTTATAAACAAGGGGGTTTTCTGGGCCTCTCCAATATTAAGTTCAGGCCAGGATCTGTGGTGGTACAATTGACTCTGGCCTTCCGAGAAGGTACCATCAATGTgCACGACGTGGAGACACAGTTCAATCAGTATAAAACGGAAGCAGCCTCTCGATATAACCTGACGATCTCAGACGTCAGCGTGAGTGATGTGCCATTTCCTTTCTCTGCCCAGTCTGGGGCTGGGGTGCCAGGCTGGGGCATCGCGCTGCTGGTGCTGGTCTGTGTTCTGGTTGCGCTGGCCATTGTCTATCTCATTGCCTTGGCTGTCTGTCAGTGCCGCCGAAAGAACTACGGGCAGCTGGACATCTTTCCAGCCCGGGATACCTACCATCCTATGAGCGAGTACCCCACCTACCACACCCATGGGCGCTATGTGCCCCCTAGCAGTACCGATCGTAGCCCCTATGAGAAGGTTTCTGCAGGTAACGGTGGCAGCAGCCTCTCTTACACAAACCCAGCAGTGGCAGCCaCTTCTGCCAA CTTGTAGcDNA sequence of UMOD C126R (mutant residue in bold and underlined)(SEQ ID NO: 2) ATGGGGCAGCCATCTCTGACTTGGATGCTGATGGTGGTGGTGGCCTCTTGGTTCATCACAACTGCAGCCACTGACACCTCAGAAGCAAGATGGTGCTCTGAATGTCACAGCAATGCCACCTGCACGGAGGATGAGGCCGTTACGACGTGCACCTGTCAGGAGGGCTTCACCGGCGATGGCCTGACCTGCGTGGACCTGGATGAGTGCGCCATTCCTGGAGCTCACAACTGCTCCGCCAACAGCAGCTGCGTAAACACGCCAGGCTCCTTCTCCTGCGTCTGCCCCGAAGGCTTCCGCCTGTCGCCCGGTCTCGGCTGCACAGACGTGGATGAGTGCGCTGAGCCTGGGCTTAGCCACTGCCACGCCCTGGCCACA C GTGTCAATGTGGTGGGCAGCTACTTGTGCGTATGCCCCGCGGGCTACCGGGGGGATGGATGGCACTGTGAGTGCTCCCCGGGCTCCTGCGGGCCGGGGTTGGACTGCGTGCCCGAGGGCGACGCGCTCGTGTGCGCGGATCCGTGCCAGGCGCACCGCACCCTGGACGAGTACTGGCGCAGCACCGAGTACGGGGAGGGCTACGCCTGCGACACGGACCTGCGCGGCTGGTACCGCTTCGTGGGCCAGGGCGGTGCGCGCATGGCCGAGACCTGCGTGCCAGTCCTGCGCTGCAACACGGCCGCCCCCATGTGGCTCAATGGCACGCATCCGTCCAGCGACGAGGGCATCGTGAGCCGCAAGGCCTGCGCGCACTGGAGCGGCCACTGCTGCCTGTGGGATGCGTCCGTCCAGGTGAAGGCCTGTGCCGGCGGCTACTACGTCTACAACCTGACAGCGCCCCCCGAGTGTCACCTGGCGTACTGCACAGACCCCAGCTCCGTGGAGGGGACGTGTGAGGAGTGCAGTATAGACGAGGACTGCAAATCGAATAATGGCAGATGGCACTGCCAGTGCAAACAGGACTTCAACATCACTGATATCTCCCTCCTGGAGCACAGGCTGGAATGTGGGGCCAATGACATGAAGGTGTCGCTGGGCAAGTGCCAGCTGAAGAGTCTGGGCTTCGACAAGGTCTTCATGTACCTGAGTGACAGCCGGTGCTCGGGCTTCAATGACAGAGACAACCGGGACTGGGTGTCTGTAGTGACCCCAGCCCGGGATGGCCCCTGTGGGACAGTGTTGACGAGGAATGAAACCCATGCCACTTACAGCAACACCCTCTACCTGGCAGATGAGATCATCATCCGTGACCTCAACATCAAAATCAACTTTGCATGCTCCTACCCCCTGGACATGAAAGTCAGCCTGAAGACCGCCCTACAGCCAATGGTCAGTGCTCTAAACATCAGAGTGGGCGGGACCGGCATGTTCACCGTGCGGATGGCGCTCTTCCAGACCCCTTCCTACACGCAGCCCTACCAAGGCTCCTCCGTGACACTGTCCACTGAGGCTTTTCTCTACGTGGGCACCATGTTGGATGGGGGCGACCTGTCCCGATTTGCACTGCTCATGACCAACTGCTATGCCACACCCAGTAGCAATGCCACGGACCCCCTGAAGTACTTCATCATCCAGGACAGATGCCCACACACTAGAGACTCAACTATCCAAGTGGTGGAGAATGGGGAGTCCTCCCAGGGCCGATTTTCCGTCCAGATGTTCCGGTTTGCTGGAAACTATGACCTAGTCTACCTGCACTGTGAAGTCTATCTCTGTGACACCATGAATGAAAAGTGCAAGCCTACCTGCTCTGGGACCAGATTCCGAAGTGGGAGTGTCATAGATCAATCCCGTGTCCTGAACTTGGGTCCCATCACACGGAAAGGTGTCCAGGCCACAGTCTCAAGGGCTTTTAGCAGCTTGGGGCTCCTGAAAGTCTGGCTGCCTCTGCTTCTCTCGGCCACCTTGACCCTGACTTTTCAGTGAcDNA sequence of rhodopsin P23H (mutant residue in bold and underlined)(SEQ ID NO: 3) AGAGTCATCCAGCTGGAGCCCTGAGTGGCTGAGCTCAGGCCTTCGCAGCATTCTTGGGTGGGAGCAGCCACGGGTCAGCCACAAGGGCCACAGCCATGAATGGCACAGAAGGCCCTAACTTCTACGTGCCCTTCTCCAATGCGACGGGTG TGGTACGCAGCC ACTTCGAGTACCCACAGTACTACCTGGCTGAGCCATGGCAGTTCTCCATGCTGGCCGCCTACATGTTTCTGCTGATCGTGCTGGGCTTCCCCATCAACTTCCTCACGCTCTACGTCACCGTCCAGCACAAGAAGCTGCGCACGCCTCTCAACTACATCCTGCTCAACCTAGCCGTGGCTGACCTCTTCATGGTCCTAGGTGGCTTCACCAGCACCCTCTACACCTCTCTGCATGGATACTTCGTCTTCGGGCCCACAGGATGCAATTTGGAGGGCTTCTTTGCCACCCTGGGCGGTGAAATTGCCCTGTGGTCCTTGGTGGTCCTGGCCATCGAGCGGTACGTGGTGGTGTGTAAGCCCATGAGCAACTTCCGCTTCGGGGAGAACCATGCCATCATGGGCGTTGCCTTCACCTGGGTCATGGCGCTGGCCTGCGCCGCACCCCCACTCGCCGGCTGGTCCAGGTACATCCCCGAGGGCCTGCAGTGCTCGTGTGGAATCGACTACTACACGCTCAAGCCGGAGGTCAACAACGAGTCTTTTGTCATCTACATGTTCGTGGTCCACTTCACCATCCCCATGATTATCATCTTTTTCTGCTATGGGCAGCTCGTCTTCACCGTCAAGGAGGCCGCTGCCCAGCAGCAGGAGTCAGCCACCACACAGAAGGCAGAGAAGGAGGTCACCCGCATGGTCATCATCATGGTCATCGCTTTCCTGATCTGCTGGGTGCCCTACGCCAGCGTGGCATTCTACATCTTCACCCACCAGGGCTCCAACTTCGGTCCCATCTTCATGACCATCCCAGCGTTCTTTGCCAAGAGCGCCGCCATCTACAACCCTGTCATCTATATCATGATGAACAAGCAGTTCCGGAACTGCATGCTCACCACCATCTGCTGCGGCAAGAACCCACTGGGTGACGATGAGGCCTCTGCTACCGTGTCCAAGACGGAGACGAGCCAGGTGGCCCCGGCCTAAGACCTGCCTAGGACTCTGTGGCCGACTATAGGCGTCTCCCATCCCCTACACCTTCCCCCAGCCACAGCCATCCCACCAGGAGCAGCGCCTGTGCAGAATGAACGAAGTCACATAGGCTCCTTAATTTTTTTTTTTTTTTTAAGAAATAATTAATGAGGCTCCTCACTCACCTGGGACAGCCTGAGAAGGGACATCCACCAAGACCTACTGATCTGGAGTCCCACGTTCCCCAAGGCCAGCGGGATGTGTGCCCCTCCTCCTCCCAACTCATCTTTCAGGAACACGAGGATTCTTGCTTTCTGGAAAAGTGTCCCAGCTTAGGGATAAGTGTCTAGCACAGAATGGGGCACACAGTAGGTGCTTAATAAATGCTGGATGGATGCAGGAAGGAATGGAGGAATGAATGGGAAGGGAGAACATATCTATCCTCTCAGACCCTCGCAGCAGCAGCAACTCATACTTGGCTAATGATATGGAGCAGTTGTTTTTCCCTCCCTGGGCCTCACTTTCTTCTCCTATAAAATGGAAATCCCAGATCCCTGGTCCTGCCGACACGCAGCTACTGAGAAGACCAAAAGAGGTGTGTGTGTGTCTATGTGTGTGTTTCAGCACTTTGTAAATAGCAAGAAGCTGTACAGATTCTAGTTAATGTTGTGAATAACATCAATTAATGTAACTAGTTAATTACTATGATTATCACCTCCTGATAGTGAACATTTTGAGATTGGGCATTCAGATGATGGGGTTTCACCCAACCTTGGGGCAGGTTTTTAAAAATTAGCTAGGCATCAAGGCCAGACCAGGGCTGGGGGTTGGGCTGTAGGCAGGGACAGTCACAGGAATGCAGAATGCAGTCATCAGACCTGAAAAAACAACACTGGGGGAGGGGGACGGTGAAGGCCAAGTTCCCAATGAGGGTGAGATTGGGCCTGGGGTCTCACCCCTAGTGTGGGGCCCCAGGTCCCGTGCCTCCCCTTCCCAATGTGGCCTATGGAGAGACAGGCCTTTCTCTCAGCCTCTGGAAGCCACCTGCTCTTTTGCTCTAGCACCTGGGTCCCAGCATCTAGAGCATGGAGCCTCTAGAAGCCATGCTCACCCGCCCACATTTAATTAACAGCTGAGTCCCTGATGTCATCCTTATCTCGAAGAGCTTAGAAACAAAGAGTGGGAAATTCCACTGGGCCTACCTTCCTTGGGGATGTTCATGGGCCCCAGTTTCCAGTTTCCCTTGCCAGACAAGCCCATCTTCAGCAGTTGCTAGTCCATTCTCCATTCTGGAGAATCTGCTCCAAAAAGCTGGCCACATCTCTGAGGTGTCAGAATTAAGCTGCCTCAGTAACTGCTCCCCCTTCTCCATATAAGCAAAGCCAGAAGCTCTAGCTTTACCCAGCTCTGCCTGGAGACTAAGGCAAATTGGGCCATTAAAAGCTCAGCTCCTATGTTGGTATTAACGGTGGTGGGTTTTGTTGCTTTCACACTCTATCCACAGGATAGATTGAAACTGCCAGCTTCCACCTGATCCCTGACCCTGGGATGGCTGGATTGAGCAATGAGCAGAGCCAAGCAGCACAGAGTCCCCTGGGGCTAGAGGTGGAGGAGGCAGTCCTG GGAATGGGAAAAACCCCATMED9 (Transmembrane P24 Trafficking Protein 9)

TMED9 is a member of a family of genes encoding transport proteinslocated in the endoplasmic reticulum and the Golgi. A representativeHomo sapiens TMED9 mRNA sequence is that of NCBI Reference SequenceNM_017510.6 (SEQ ID NO: 4):

AGGTGGAGCAAGATGGCTGTGGAGCTGGGCGTGCTGCTCGTCCGGCCCCGGCCCGGAACCGGGCTGGGTAGAGTGATGCGGACCCTCCTGCTGGTGCTGTGGCTGGCGACGCGCGGAAGCGCGCTCTACTTTCACATCGGAGAGACGGAGAAGAAGTGCTTTATTGAGGAGATCCCGGACGAGACCATGGTCATAGGAAACTACCGGACGCAGCTGTATGACAAGCAGCGGGAGGAGTACCAGCCGGCCACCCCGGGGCTTGGCATGTTTGTGGAGGTGAAGGACCCAGAGGACAAGGTCATCCTGGCCCGGCAGTATGGCTCCGAGGGCAGGTTCACTTTCACTTCCCATACCCCTGGTGAGCACCAGATCTGTCTTCACTCCAATTCCACCAAGTTCTCCCTCTTTGCTGGAGGCATGCTGAGAGTTCACCTGGACATCCAGGTAGGTGAACATGCCAATGACTATGCAGAAATTGCTGCTAAAGACAAGTTGAGTGAGTTGCAGCTACGAGTGCGACAGCTGGTGGAACAAGTGGAGCAGATCCAGAAAGAGCAGAACTACCAGCGGTGGCGAGAGGAGCGCTTCCGGCAGACCAGTGAGAGCACCAACCAGCGGGTGCTGTGGTGGTCCATTCTGCAGACCCTCATCCTCGTGGCCATCGGTGTCTGGCAGATGCGGCACCTCAAGAGCTTCTTTGAAGCCAAGAAGCTTGTGTAGCTGTCCCAGGCGTCACAACCCATCCTCCCAGGCTGGGGGAGAAAGGACCTCCTGGAACTGACTTCTTCTGTCAGGAGGACTGGTTTCCAGCCATACCTGTTCTGGAAGGGAGAGGGGCTGGAGGCACCCACAGGCACAAGCTGAAGGCAGCAGCTTGGCTAATACTGAGCAGGTAGTGGGGCAAATTCCTGCCCTCTCTCTCTGGCCTCTGGGCCGTTTGGTAGTAATCACCCAAGGGCTGGTAAAGCCCCTCCTCTTGGCACCTCAGAATCACAGTGTTACTGATCAGGGATGTGAGGCTGCTGTTGGGGGTGGGGGGAGGGGAATGGGCAGGCAAGCCAGTCTTCTGTCTTCCTTTGCTAACTTAGGGTTTTGAGCAGGTTGGGGTATGGTGCCTGTCATACCCACCTGCCACCCTGGGAACCTCACTGTTCTCTCTTTCAGCCTAGACCTGCTGATCCAGGGTGTGTGTGAGTTGAGGGTGGGTGGAGGGGTTTGCAGTGTGGGAATGTGGCCCTGCAGTTGACCTGAGCTGCTTCACATGGTTGTCCATTCTGGGGCTTAAAGAACTGGGACCAGACCAAGTAGAGGCCTTGGTGCTGGTTGGGGTGGGGCCTGCAGAGTCTTAGTTACTGATTTCATTTTCAATAAATGTAGGTTTGTTACATGAGTTTCCCAATAAAAAAAAAAATGACTTCTTGTCCAGTGCAAGTGACTCAGTCATCAGTGGGCACACACTGCAGGGTGCCTCAGGGAATGCCAGTTCTTCCAAAGAGCAAAGCACTTCACATTCCAAAGTGAATTCCCACCAGTCAGCTTCATTCTTTCCTTCTTCTCCAGGCCTTCCTGTGGCAGGGAATAGTGGGTTTGTCCAAGATTATACAACAAGTAAATTGGGCTGGGGCTCAAATTTACACCCTTTCCTCTGTGCCAGCTCCCTGGTGAAGTTCCCTCTTTCTAGAGTCAGTAAGCAGGATTGTCATGGATGCTGCCAGGAAGTGCCTGGTAAGGAGGTGCATTGAGCAGGGGAGTGCTACAGGACAGCCACCCTGGGCTGGCAGGGACAAGGATGTTGATGGGCTAAACCAACAGCAAGTGATTTCAACCAGGACCATGAAGGAGAGGAAGGATTCTGCTGGAAGGAGATGGCAGGACAGGGGTGGTTGGAGAAGTGGAGGCAAACAGCTGGAATGGAGGTGGGTGGGTGTTTAATTTCAGCTGCAGAGGGTGTTGTGAGGAAGCTGGAAAGGAAGGTTGGATTAGAGAAGCCTCGAGCTCCAGGTAAGCGATTTGGACATGCCCACCTTTCAAGAGGGGCTGCAGGCACCCACAGGCACAAGCTGAAGGCAGCAGCTTGGCTGGCTTAATACTGAGCAGGTGGTGGGGTAAATGCCTGCCCCCCTCCCTCTGGCCTCTGGGCCCTTTGCAGTAATCACCCAGGGTCTGGTAAAGCCACTGAGAGCCCTACTGGCACCTCAGAATCACAGTGTTATTGATCAGGGATGTGAGGCTGCTGTTGGGGGTTGGGGGAGGCAAATGGGCAGGCAGTTTTGAGAAGAACCTTCTAATAAGAAATGTGAGGGAGGTTACAGCAGTGTGTGAGAAAGACCAGGAAGAAGGAGACAAGTTTGGGGGCTGCTTCCCCTAATGGGATGATGCAATCTGGGCTCATGCTGCCAACTAATTCTTCCACATGAAAAAAAAAAGTTTTTTGGCGGGCACGGTGGTTCACACCTGTAATCCCAGCACTTTGGGAGGCTGAGGTAGGTGGATGGCCTGAGGCCAGGAGTTTGAGACCAGCCTGGCCAACATGGTGAAACCTCATC

A corresponding representative Homo sapiens TMED9 protein sequence isthat of (SEQ ID NO: 5):

MAVELGVLLVRPRPGTGLGRVMRTLLLVLWLATRGSALYFHIGETEKKCFIEEIPDETMVIGNYRTQLYDKQREEYQPATPGLGMFVEVKDPEDKVILARQYGSEGRFTFTSHTPGEHQICLHSNSTKFSLFAGGMLRVHLDIQVGEHANDYAEIAAKDKLSELQLRVRQLVEQVEQIQKEQNYQRWREERFRQTSESTNQRVLWWSILQTLILVAIGVWQMRHLKSFFEAKKLVTreatment Selection

The compositions and methods described herein can be used for selecting,and then optionally administering, an optimal treatment (e.g., aTMED9-binding agent, e.g., BRD-4780, alone (as a mixture of enantiomers(racemic or non-racemic), or as one enantiomer) or in combination withother agents). Generally, the methods include administering atherapeutically effective amount of a treatment as described herein, toa subject who is in need of, or who has been determined to be in needof, such treatment.

As used in this context, to “treat” means to ameliorate at least onesymptom of the proteinopathy. For example, a treatment can result inimproved kidney function and/or amelioration in the rate of decline ofkidney function that would occur in the absence of treatment, improvedneurodegenerative disease and/or eye functions and/or amelioration inthe rate of neurodegeneration and/or the rate of declining eye functionin a subject having or at risk of a toxic proteinopathy resulting frommutant protein accumulation in the early secretory pathway, or in otherorganelles of the secretory pathway.

Exemplary neurodegenerative diseases of the instant disclosure include,without limitation, Alzheimer's disease (AD) and other dementias;Parkinson's disease (PD) and PD-related disorders; prion disease(including, e.g., Creutzfeldt-Jakob Disease, variant Creutzfeldt-JakobDisease, Bovine Spongiform Encephalopathy, Kuru,Gerstmann-Straussler-Scheinker disease, fatal familial insomnia (FFI),scrapie, and other animal TSEs); motor neuron diseases (MND; including,e.g., Amyotrophic Lateral Sclerosis (ALS), Primary Lateral Sclerosis(PLS), Progressive Bulbar Palsy (PBP), Pseudobulbar Palsy, ProgressiveMuscular Atrophy, Spinal Muscular Atrophy (Type 1, Type 2, Type 3, Type4), and Kennedy's Disease); and spinocerebellar ataxia (SCA).

In certain embodiments, the methods of the instant disclosure caninclude selecting and/or administering a treatment that includes atherapeutically effective amount of a TMED9-binding agent, e.g.,BRD-4780. A TMED9-binding agent (e.g., BRD-4780) of the instantdisclosure may be administered alone to a subject, or, optionally, theTMED9-binding agent (e.g., BRD-4780) may be administered in combinationwith an additional therapeutic agent. Without limitation, specificallycontemplated combination therapies for MKD include administration of aTMED9-binding agent, e.g., BRD-4780, and any of the following: vitamin Din any or all of its forms (e.g., ergocalciferol, cholecalciferol,others), phosphate binders, blood pressure medications and diuretics.Specific examples of phosphate binders, blood pressure medications anddiuretics include the following, with exemplary dosages also indicated:

Phosphate Binders:

Calcium Acetate (PhosLo, Calphron, Eliphos, PhosLo Gelcap, andPhoslyra)—667 mg or 667 mg in 5 ml (oral solution)

Sevelamer (Renagel and Renvela)—400 mg (Renagel tablet), 800 mg (Renageltablet, Renvela tablet, and Renvela powder packet), and 2400 mg (Renvelapowder packet)

Ferric Citrate (Auryxia)—210 mg (tablet)

Lanthanum Carbonate (Fosrenol)—Tablet (500 mg, 750 mg, and 1000 mg) andOral powder (750 mg and 1000 mg).

Sucroferric Oxyhydroxide (Velphoro)—500 mg

Aluminum Hydroxide (Amphojel)—320 mg/5 ml oral suspension

Diuretics:

Bumetanide (Bumex)—0.5 mg (light green), 1 mg (yellow) and 2 mg (peach)tablets for oral administration.

Chlorthalidone (Thalitone)—Oral Tablet: 15 mg, 25 mg, 50 mg

Chlorothiazide (Diuril)—Adults (500 or 1000 mg IV/Tablet)

Ethacrynate (Edecrin)—25 mg tablets for oral use

Furosemide (Lasix)—Tablets 20, 40, and 80 mg

Hydrochlorothiazide HCTZ (Esidrix, Hydrodiuril, Microzide)—25 mg; 50 mg;100 mg; 50 mg/5 mL; 12.5 mg

Indapamide (Lozol)—2.5 mg orally once a day.

Methyclothiazide (Enduron)—2.5 to 5 mg orally once a day

Metolazone (Mykroz, Zaroxolyn)—2.5 mg orally once a day (Zaroxolyn) or

0.5 mg orally once a day (Mykrox).

Torsemide (Demadex)—5 mg orally once a day; if diuresis remainsinadequate after 4 to 6 weeks, titrate up to 10 mg orally once a day; ifdiuresis remains inadequate with 10 mg, an additional antihypertensiveis added.

Blood Pressure Medications:

Beta Blockers:

acebutolol (Sectral)—200 mg or 400 mg tablet

atenolol (Tenormin)—25 mg, 50 mg, and 100 mg tablet.

betaxolol (Kerlone)—10 mg or 20 mg.

bisoprolol (Zebeta)—5 mg or 10 mg tablet.

bisoprolol/hydrochlorothiazide (Ziac)—Bisprolol (2.5 mg)/hydrochloride(6.25 mg) tablet.

metoprolol tartrate (Lopressor)—100 mg tablet daily

metoprolol succinate (Toprol-XL)—25 mg to 100 mg daily.

nadolol (Corgard)—40 mg tablet daily.

pindolol (Visken)—5 mg tablet initial dose.

propranolol (Inderal)—40 mg twice daily.

solotol (Betapace)—80 mg-240 mg tablets in 80 mg increments.

timolol (Blocadren)—5 mg, 10 mg, and 20 mg tablet.

ACE Inhibitors:

benazepril (Lotensin)—10 mg, 20 mg, and 40 mg tablets.

captopril (Capoten)—12.5 mg, 25 mg, 50 mg, and 100 mg tablet.

enalapril (Vasotec)—5 mg initial daily dose.

fosinopril (Monopril)—10 mg once a day

lisinopril (Prinivil, Zestril)—2.5 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mgtablets 1 mg/ml oral solution.

moexipril (Univasc)—7.5 mg and 15 mg tablets.

perindopril (Aceon)—2 mg, 4 mg, and 8 mg tablets.

quinapril (Accupril)—5 mg, 10 mg, 20 mg, and 40 mg tablets.

-   -   ramipril (Altace)—1.25 mg tablet, 2.5 mg, and 5 mg tablet.    -   trandolapril (Mavik)—1 mg, 2 mg, and 4 mg tablet.

Calcium Channel Blockers

-   -   amlodipine (Norvasc, Lotrel)—10 mg orally    -   diltiazem (Cardizem CD, Cardizem SR, Dilacor XR, Tiazac)—20 mg        average adult dose    -   felodipine (Plendil)—2.5 mg, 5 mg, and 10 mg oral tablet.    -   isradipine (DynaCirc, DynaCirc CR)—7.5 mg daily    -   nicardipine (Cardene SR)—20 mg and 30 mg capsule.    -   nifedipine (Adalat CC, Procardia XL)—10 mg, 20 mg, 30 mg, 60 mg,        and 90 mg tablet.    -   nisoldipine (Sular)—17 mg orally daily.    -   verapamil (Calan SR, Covera HS, Isoptin SR, Verelan)—100 mg/200        mg daily.

Alpha Blockers

doxazosin (Cardura)—2 mg, 4 mg, and 8 mg daily.

prazosin (Minipress)—20 mg total daily dose.

terazosin (Hytrin)—1 mg at bedtime

Alpha-Beta-Blockers

-   -   carvedilol (Coreg)—3.125 mg, 6.25 mg, 12.5 mg, and 25 mg tablet.    -   labetalol (Normodyne, Trandate)—100 mg, 200 mg, and 300 mg taken        orally.

Central Agonists

-   -   methyldopa (Aldomet)—125 mg, 250 mg, and 500 mg tablet.    -   clonidine (Catapres)—0.1 mg, 0.2 mg, and 0.3 mg.    -   guanfacine (Tenex)—1 mg, 2 mg, 3 mg, and 4 mg.

Vasodilators

hydralazine (Apresoline)—25 mg, 50 mg, 10 mg, 100 mg, and 20 mg/ml.

-   -   minoxidil (Loniten)—2.5 mg and 10 mg tablet.

Without wishing to be bound by theory, though BRD-4780 has beenprimarily exemplified for its effect upon the early secretory pathway,it is contemplated that actions of BRD-4780 (or other TMED9-bindingagents) upon the late secretory pathway could also exert a beneficialeffect. Thus, it is contemplated that the compositions and methods ofthe instant disclosure could also address proteinopathy and relatedeffects in organelles of the late secretory pathway including, withoutlimitation, post-Golgi trafficking vesicles (whether directed to theendosome, including, e.g., ESCRT-II complex vesicles, and/orendosome-bypassing lysosomal transport vesicles and/or cellsurface-directed vesicles), the endosome, and/or post-endosomaltransport vesicles, including, without limitation, endosome-to-lysosomevesicles, endosome-to-cell surface transport vesicles (including, e.g.,synaptic vesicles) and cell surface-to-endosome vesicles, and thelysosome.

The structure of BRD-4780 follows.

BRD-4780

2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane (identified asBRD-4780 herein and also known as (±) AGN 192403,2-propan-2-ylbicyclo[2.2.1]heptan-3-amine,3-Isopropylbicyclo[2.2.1]heptan-2-amine, andtrans-2-(3-Isopropyl-bicyclo-[2,2,1]-heptyl)-amine), has the followingstructure of formula (II):

2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane is commerciallyavailable as a racemic hydrochloride salt.

As exemplified herein, synthesis and separation of each of theenantiomers of racemic 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptanehas now been performed. It is explicitly contemplated that individualenantiomeric forms of 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptanecan be employed in any manner that is currently described for BRD-4780,also including, without limitation, compositions and use ofpharmaceutically acceptable salts of such single enantiomer forms, e.g.,including hydrochloride salt forms of individual enantiomers of2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane.

2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane was disclosed inPCT/US1995/008733 (Publication No. WO 96/01813-related patents include:U.S. Pat. Nos. 5,731,337; 6,150,389; 6,319,935; and 6,706,747), as werethe following structures:

Combination Treatments

The compositions and methods of the present disclosure may be used inthe context of a number of therapeutic or prophylactic applications. Inorder to increase the effectiveness of a treatment with the compositionsof the present disclosure, e.g., TMED9-binding agents, e.g., BRD-4780selected and/or administered as a single agent, can be selected and/oradministered with another agent or therapy, optionally to augment theefficacy of another therapy (second therapy). Thus, it may be desirableto combine these compositions and methods with one another, or withother agents and methods effective in the treatment, amelioration, orprevention of diseases and pathologic conditions, for example, toxicproteinopathies resulting from mutant protein accumulation in the earlysecretory pathway, such as a neurodegenerative disease, MKD, anautosomal dominant kidney disease caused by uromodulin mutation, a formof retinitis pigmentosa caused by rhodopsin mutation, etc.

Administration of a composition of the present disclosure to a subjectwill follow general protocols for the administration described herein,and the general protocols for the administration of a particularsecondary therapy will also be followed, taking into account thetoxicity, if any, of the treatment. It is expected that the treatmentcycles would be repeated as necessary. It also is contemplated thatvarious standard therapies may be applied in combination with thedescribed therapies.

Pharmaceutical Compositions

Agents of the present disclosure can be incorporated into a variety offormulations for therapeutic use (e.g., by administration) or in themanufacture of a medicament (e.g., for treating or preventing aproteinopathy) by combining the agents with appropriate pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms. Examples of suchformulations include, without limitation, tablets, capsules, powders,granules, ointments, solutions, suppositories, injections, inhalants,gels, microspheres, and aerosols.

Pharmaceutical compositions can include, depending on the formulationdesired, pharmaceutically-acceptable, non-toxic carriers of diluents,which are vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents include, without limitation, distilled water, bufferedwater, physiological saline, PBS, Ringer's solution, dextrose solution,and Hank's solution. A pharmaceutical composition or formulation of thepresent disclosure can further include other carriers, adjuvants, ornon-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients andthe like. The compositions can also include additional substances toapproximate physiological conditions, such as pH adjusting and bufferingagents, toxicity adjusting agents, wetting agents and detergents.

Further examples of formulations that are suitable for various types ofadministration can be found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985). For a briefreview of methods for drug delivery, see, Langer, Science 249: 1527-1533(1990).

For oral administration, the active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. The activecomponent(s) can be encapsulated in gelatin capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.Examples of additional inactive ingredients that may be added to providedesirable color, taste, stability, buffering capacity, dispersion orother known desirable features are red iron oxide, silica gel, sodiumlauryl sulfate, titanium dioxide, and edible white ink.

Similar diluents can be used to make compressed tablets. Both tabletsand capsules can be manufactured as sustained release products toprovide for continuous release of medication over a period of hours.Compressed tablets can be sugar coated or film coated to mask anyunpleasant taste and protect the tablet from the atmosphere, orenteric-coated for selective disintegration in the gastrointestinaltract. Liquid dosage forms for oral administration can contain coloringand flavoring to increase patient acceptance.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts of amines, carboxylic acids, and other types ofcompounds, are well known in the art. For example, S. M. Berge, et al.describe pharmaceutically acceptable salts in detail in J PharmaceuticalSciences 66 (1977):1-19, incorporated herein by reference. The salts canbe prepared in situ during the final isolation and purification of thecompounds of the application, or separately by reacting a free base orfree acid function with a suitable reagent, as described generallybelow. For example, a free base function can be reacted with a suitableacid. Furthermore, where the compounds to be administered of theapplication carry an acidic moiety, suitable pharmaceutically acceptablesalts thereof may, include metal salts such as alkali metal salts, e.g.sodium or potassium salts; and alkaline earth metal salts, e.g. calciumor magnesium salts. Examples of pharmaceutically acceptable, nontoxicacid addition salts are salts of an amino group formed with inorganicacids such as hydrochloric acid, hydrobromic acid, phosphoric acid,sulfuric acid and perchloric acid or with organic acids such as aceticacid, oxalic acid, maleic acid, tartaric acid, citric acid, succinicacid or malonic acid or by using other methods used in the art such asion exchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, lower alkyl sulfonate and aryl sulfonate.

Additionally, as used herein, the term “pharmaceutically acceptableester” refers to esters that hydrolyze in vivo and include those thatbreak down readily in the human body to leave the parent compound (e.g.,an FDA-approved compound where administered to a human subject) or asalt thereof. Suitable ester groups include, for example, those derivedfrom pharmaceutically acceptable aliphatic carboxylic acids,particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, inwhich each alkyl or alkenyl moeity advantageously has not more than 6carbon atoms. Examples of particular esters include formates, acetates,propionates, butyrates, acrylates and ethylsuccinates.

Furthermore, the term “pharmaceutically acceptable prodrugs” as usedherein refers to those prodrugs of the certain compounds of the presentapplication which are, within the scope of sound medical judgment,suitable for use in contact with the issues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the application. The term “prodrug” refers tocompounds that are rapidly transformed in vivo to yield the parentcompound of an agent of the instant disclosure, for example byhydrolysis in blood. A thorough discussion is provided in T. Higuchi andV. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S.Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers inDrug Design, American Pharmaceutical Association and Pergamon Press,(1987), both of which are incorporated herein by reference.

The components used to formulate the pharmaceutical compositions arepreferably of high purity and are substantially free of potentiallyharmful contaminants (e.g., at least National Food (NF) grade, generallyat least analytical grade, and more typically at least pharmaceuticalgrade). Moreover, compositions intended for in vivo use are usuallysterile. To the extent that a given compound must be synthesized priorto use, the resulting product is typically substantially free of anypotentially toxic agents, particularly any endotoxins, which may bepresent during the synthesis or purification process. Compositions forparental administration are also sterile, substantially isotonic andmade under GMP conditions.

Formulations may be optimized for retention and stabilization in asubject and/or tissue of a subject, e.g., to prevent rapid clearance ofa formulation by the subject. Stabilization techniques includecross-linking, multimerizing, or linking to groups such as polyethyleneglycol, polyacrylamide, neutral protein carriers, etc. in order toachieve an increase in molecular weight.

Other strategies for increasing retention include the entrapment of theagent, such as a TMED9-binding agent (e.g., BRD-4780), in abiodegradable or bioerodible implant. The rate of release of thetherapeutically active agent is controlled by the rate of transportthrough the polymeric matrix, and the biodegradation of the implant. Thetransport of drug through the polymer barrier will also be affected bycompound solubility, polymer hydrophilicity, extent of polymercross-linking, expansion of the polymer upon water absorption so as tomake the polymer barrier more permeable to the drug, geometry of theimplant, and the like. The implants are of dimensions commensurate withthe size and shape of the region selected as the site of implantation.Implants may be particles, sheets, patches, plaques, fibers,microcapsules and the like and may be of any size or shape compatiblewith the selected site of insertion.

The implants may be monolithic, i.e. having the active agenthomogenously distributed through the polymeric matrix, or encapsulated,where a reservoir of active agent is encapsulated by the polymericmatrix. The selection of the polymeric composition to be employed willvary with the site of administration, the desired period of treatment,patient tolerance, the nature of the disease to be treated and the like.Characteristics of the polymers will include biodegradability at thesite of implantation, compatibility with the agent of interest, ease ofencapsulation, a half-life in the physiological environment.

Biodegradable polymeric compositions which may be employed may beorganic esters or ethers, which when degraded result in physiologicallyacceptable degradation products, including the monomers. Anhydrides,amides, orthoesters or the like, by themselves or in combination withother monomers, may find use. The polymers will be condensationpolymers. The polymers may be cross-linked or non-cross-linked. Ofparticular interest are polymers of hydroxyaliphatic carboxylic acids,either homo- or copolymers, and polysaccharides. Included among thepolyesters of interest are polymers of D-lactic acid, L-lactic acid,racemic lactic acid, glycolic acid, polycaprolactone, and combinationsthereof. By employing the L-lactate or D-lactate, a slowly biodegradingpolymer is achieved, while degradation is substantially enhanced withthe racemate. Copolymers of glycolic and lactic acid are of particularinterest, where the rate of biodegradation is controlled by the ratio ofglycolic to lactic acid. The most rapidly degraded copolymer has roughlyequal amounts of glycolic and lactic acid, where either homopolymer ismore resistant to degradation. The ratio of glycolic acid to lactic acidwill also affect the brittleness of in the implant, where a moreflexible implant is desirable for larger geometries. Among thepolysaccharides of interest are calcium alginate, and functionalizedcelluloses, particularly carboxymethylcellulose esters characterized bybeing water insoluble, a molecular weight of about 5 kD to 500 kD, etc.Biodegradable hydrogels may also be employed in the implants of theindividual instant disclosure. Hydrogels are typically a copolymermaterial, characterized by the ability to imbibe a liquid. Exemplarybiodegradable hydrogels which may be employed are described in Hellerin: Hydrogels in Medicine and Pharmacy, N. A. Peppes ed., Vol. III, CRCPress, Boca Raton, Fla., 1987, pp 137-149.

Pharmaceutical Dosages

Pharmaceutical compositions of the present disclosure containing anagent described herein may be used (e.g., administered to an individual,such as a human individual, in need of treatment with a TMED9-bindingagent (e.g., BRD-4780)) in accord with known methods, such as oraladministration, intravenous administration as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerobrospinal, intracranial, intraspinal, subcutaneous,intraarticular, intrasynovial, intrathecal, topical, or inhalationroutes.

Dosages and desired drug concentration of pharmaceutical compositions ofthe present disclosure may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary artisan. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles described in Mordenti, J. andChappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” InToxicokinetics and New Drug Development, Yacobi et al., Eds, PergamonPress, New York 1989, pp. 42-46.

For in vivo administration of any of the agents of the presentdisclosure, normal dosage amounts may vary from about 10 ng/kg up toabout 100 mg/kg of an individual's and/or subject's body weight or moreper day, depending upon the route of administration. In someembodiments, the dose amount is about 1 mg/kg/day to 10 mg/kg/day. Forrepeated administrations over several days or longer, depending on theseverity of the disease, disorder, or condition to be treated, thetreatment is sustained until a desired suppression of symptoms isachieved.

An effective amount of an agent of the instant disclosure may vary,e.g., from about 0.001 mg/kg to about 1000 mg/kg or more in one or moredose administrations for one or several days (depending on the mode ofadministration). In certain embodiments, the effective amount per dosevaries from about 0.001 mg/kg to about 1000 mg/kg, from about 0.01 mg/kgto about 750 mg/kg, from about 0.1 mg/kg to about 500 mg/kg, from about1.0 mg/kg to about 250 mg/kg, and from about 10.0 mg/kg to about 150mg/kg.

An exemplary dosing regimen may include administering an initial dose ofan agent of the disclosure of about 200 μg/kg, followed by a weeklymaintenance dose of about 100 μg/kg every other week. Other dosageregimens may be useful, depending on the pattern of pharmacokineticdecay that the physician wishes to achieve. For example, dosing anindividual from one to twenty-one times a week is contemplated herein.In certain embodiments, dosing ranging from about 3 μg/kg to about 2mg/kg (such as about 3 μg/kg, about 10 μg/kg, about 30 μg/kg, about 100μg/kg, about 300 μg/kg, about 1 mg/kg, or about 2 mg/kg) may be used. Incertain embodiments, dosing frequency is three times per day, twice perday, once per day, once every other day, once weekly, once every twoweeks, once every four weeks, once every five weeks, once every sixweeks, once every seven weeks, once every eight weeks, once every nineweeks, once every ten weeks, or once monthly, once every two months,once every three months, or longer. Progress of the therapy is easilymonitored by conventional techniques and assays. The dosing regimen,including the agent(s) administered, can vary over time independently ofthe dose used.

Pharmaceutical compositions described herein can be prepared by anymethod known in the art of pharmacology. In general, such preparatorymethods include the steps of bringing the agent or compound describedherein (i.e., the “active ingredient”) into association with a carrieror excipient, and/or one or more other accessory ingredients, and then,if necessary and/or desirable, shaping, and/or packaging the productinto a desired single- or multi-dose unit.

Pharmaceutical compositions can be prepared, packaged, and/or sold inbulk, as a single unit dose, and/or as a plurality of single unit doses.A “unit dose” is a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient. The amountof the active ingredient is generally equal to the dosage of the activeingredient which would be administered to a subject and/or a convenientfraction of such a dosage such as, for example, one-half or one-third ofsuch a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition described herein will vary, depending uponthe identity, size, and/or condition of the subject treated and furtherdepending upon the route by which the composition is to be administered.The composition may comprise between 0.1% and 100% (w/w) activeingredient.

Pharmaceutically acceptable excipients used in the manufacture ofprovided pharmaceutical compositions include inert diluents, dispersingand/or granulating agents, surface active agents and/or emulsifiers,disintegrating agents, binding agents, preservatives, buffering agents,lubricating agents, and/or oils. Excipients such as cocoa butter andsuppository waxes, coloring agents, coating agents, sweetening,flavoring, and perfuming agents may also be present in the composition.

Exemplary diluents include calcium carbonate, sodium carbonate, calciumphosphate, dicalcium phosphate, calcium sulfate, calcium hydrogenphosphate, sodium phosphate lactose, sucrose, cellulose,microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodiumchloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

Exemplary granulating and/or dispersing agents include potato starch,corn starch, tapioca starch, sodium starch glycolate, clays, alginicacid, guar gum, citrus pulp, agar, bentonite, cellulose, and woodproducts, natural sponge, cation-exchange resins, calcium carbonate,silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone)(crospovidone), sodium carboxymethyl starch (sodium starch glycolate),carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose(croscarmellose), methylcellulose, pregelatinized starch (starch 1500),microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate,quaternary ammonium compounds, and mixtures thereof.

Exemplary surface active agents and/or emulsifiers include naturalemulsifiers (e.g., acacia, agar, alginic acid, sodium alginate,tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk,casein, wool fat, cholesterol, wax, and lecithin), colloidal clays(e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminumsilicate)), long chain amino acid derivatives, high molecular weightalcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetinmonostearate, ethylene glycol distearate, glyceryl monostearate, andpropylene glycol monostearate, polyvinyl alcohol), carbomers (e.g.,carboxy polymethylene, polyacrylic acid, acrylic acid polymer, andcarboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.,carboxymethylcellulose sodium, powdered cellulose, hydroxy methylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylenesorbitan monolaurate (Tween® 20), polyoxyethylene sorbitan (Tween® 60),polyoxyethylene sorbitan monooleate (Tween® 80), sorbitan monopalmitate(Span® 40), sorbitan monostearate (Span® 60), sorbitan tristearate(Span® 65), glyceryl monooleate, sorbitan monooleate (Span® 80),polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj® 45),polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g., Cremophor®),polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij® 30)),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, Pluronic® F-68, Poloxamer P-188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, and/or mixtures thereof.

Exemplary binding agents include starch (e.g., cornstarch and starchpaste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin,molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums(e.g., acacia, sodium alginate, extract of Irish moss, panwar gum,ghatti gum, mucilage of isapol husks, carboxymethylcellulose,methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, microcrystalline cellulose,cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate(Veegum®), and larch arabogalactan), alginates, polyethylene oxide,polyethylene glycol, inorganic calcium salts, silicic acid,polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.

Exemplary preservatives include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, antiprotozoanpreservatives, alcohol preservatives, acidic preservatives, and otherpreservatives. In certain embodiments, the preservative is anantioxidant. In other embodiments, the preservative is a chelatingagent.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene,monothioglycerol, potassium metabisulfite, propionic acid, propylgallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, andsodium sulfite.

Exemplary chelating agents include ethylenediaminetetraacetic acid(EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodiumedetate, trisodium edetate, calcium disodium edetate, dipotassiumedetate, and the like), citric acid and salts and hydrates thereof(e.g., citric acid monohydrate), fumaric acid and salts and hydratesthereof, malic acid and salts and hydrates thereof, phosphoric acid andsalts and hydrates thereof, and tartaric acid and salts and hydratesthereof. Exemplary antimicrobial preservatives include benzalkoniumchloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide,cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol,chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea,phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate,propylene glycol, and thimerosal.

Exemplary antifungal preservatives include butyl paraben, methylparaben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoicacid, potassium benzoate, potassium sorbate, sodium benzoate, sodiumpropionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol,phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxy benzoate,and phenylethyl alcohol.

Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E,beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbicacid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, deteroximemesylate, cetrimide, butylated hydroxyanisol (BHA), butylatedhydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS),sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, Glydant®Plus, Phenonip®, methylparaben, German® 115, Germaben® II, Neolone®,Kathon®, and Euxyl®.

Exemplary buffering agents include citrate buffer solutions, acetatebuffer solutions, phosphate buffer solutions, ammonium chloride, calciumcarbonate, calcium chloride, calcium citrate, calcium glubionate,calcium gluceptate, calcium gluconate, D-gluconic acid, calciumglycerophosphate, calcium lactate, propanoic acid, calcium levulinate,pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasiccalcium phosphate, calcium hydroxide phosphate, potassium acetate,potassium chloride, potassium gluconate, potassium mixtures, dibasicpotassium phosphate, monobasic potassium phosphate, potassium phosphatemixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodiumcitrate, sodium lactate, dibasic sodium phosphate, monobasic sodiumphosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide,aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline,Ringer's solution, ethyl alcohol, and mixtures thereof.

Exemplary lubricating agents include magnesium stearate, calciumstearate, stearic acid, silica, talc, malt, glyceryl behanate,hydrogenated vegetable oils, polyethylene glycol, sodium benzoate,sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate,sodium lauryl sulfate, and mixtures thereof.

Exemplary natural oils include almond, apricot kernel, avocado, babassu,bergamot, black current seed, borage, cade, camomile, canola, caraway,camauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee,corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed,geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate,jojoba, kukui nut, lavandin, lavender, lemon, Litsea cubeba, macademianut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,orange roughy, palm, palm kernel, peach kemel, peanut, poppy seed,pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, andwheat germ oils. Exemplary synthetic oils include, but are not limitedto, butyl stearate, caprylic triglyceride, capric triglyceride,cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate,mineral oil, octyldodecanol, ° leyl alcohol, silicone oil, and mixturesthereof.

Liquid dosage forms for oral and parenteral administration includepharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active ingredients,the liquid dosage forms may comprise inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can include adjuvants such as wetting agents, emulsifyingand suspending agents, sweetening, flavoring, and perfuming agents. Incertain embodiments for parenteral administration, the conjugatesdescribed herein are mixed with solubilizing agents such as Cremophor®,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and mixtures thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation can be a sterile injectable solution,suspension, or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution, U.S.P., and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or di-glycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This can be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform may be accomplished by dissolving or suspending the drug in an oilvehicle.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing the conjugates describedherein with suitable non-irritating excipients or carriers such as cocoabutter, polyethylene glycol, or a suppository wax which are solid atambient temperature but liquid at body temperature and therefore melt inthe rectum or vaginal cavity and release the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or (a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, (b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, (c) humectants such as glycerol, (d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, (e) solutionretarding agents such as paraffin, (f) absorption accelerators such asquaternary ammonium compounds, (g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolinand bentonite clay, and (i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets, and pills, thedosage form may include a buffering agent.

Solid compositions of a similar type can be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the art of pharmacology. Theymay optionally comprise opacifying agents and can be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of encapsulating compositions which can be used includepolymeric substances and waxes. Solid compositions of a similar type canbe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugar as well as high molecularweight polethylene glycols and the like.

The active ingredient can be in a micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings, and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active ingredient can be admixed with at least oneinert diluent such as sucrose, lactose, or starch. Such dosage forms maycomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such asmagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may comprise bufferingagents. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of encapsulating agents which can be usedinclude polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of an agent(e.g., a TMED9-binding agent, e.g., BRD-4780) described herein mayinclude ointments, pastes, creams, lotions, gels, powders, solutions,sprays, inhalants, and/or patches. Generally, the active ingredient isadmixed under sterile conditions with a pharmaceutically acceptablecarrier or excipient and/or any needed preservatives and/or buffers ascan be required. Additionally, the present disclosure contemplates theuse of transdermal patches, which often have the added advantage ofproviding controlled delivery of an active ingredient to the body. Suchdosage forms can be prepared, for example, by dissolving and/ordispensing the active ingredient in the proper medium. Alternatively oradditionally, the rate can be controlled by either providing a ratecontrolling membrane and/or by dispersing the active ingredient in apolymer matrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices. Intradermalcompositions can be administered by devices which limit the effectivepenetration length of a needle into the skin. Alternatively oradditionally, conventional syringes can be used in the classical mantouxmethod of intradermal administration. Jet injection devices whichdeliver liquid formulations to the dermis via a liquid jet injectorand/or via a needle which pierces the stratum comeum and produces a jetwhich reaches the dermis are suitable. Ballistic powder/particledelivery devices which use compressed gas to accelerate the compound inpowder form through the outer layers of the skin to the dermis aresuitable.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi-liquid preparations such as liniments,lotions, oil-in-water and/or water-in-oil emulsions such as creams,ointments, and/or pastes, and/or solutions and/or suspensions. Topicallyadministrable formulations may, for example, comprise from about 1% toabout 10% (w/w) active ingredient, although the concentration of theactive ingredient can be as high as the solubility limit of the activeingredient in the solvent. Formulations for topical administration mayfurther comprise one or more of the additional ingredients describedherein.

A pharmaceutical composition described herein can be prepared, packaged,and/or sold in a formulation suitable for pulmonary administration viathe buccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers, or from about 1 to about 6nanometers. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant can be directed to dispersethe powder and/or using a self-propelling solvent/powder dispensingcontainer such as a device comprising the active ingredient dissolvedand/or suspended in a low-boiling propellant in a sealed container. Suchpowders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositions mayinclude a solid fine powder diluent such as sugar and are convenientlyprovided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic and/or solid anionic surfactant and/or a solid diluent(which may have a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions described herein formulated for pulmonarydelivery may provide the active ingredient in the form of droplets of asolution and/or suspension. Such formulations can be prepared, packaged,and/or sold as aqueous and/or dilute alcoholic solutions and/orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization and/oratomization device. Such formulations may further comprise one or moreadditional ingredients including, but not limited to, a flavoring agentsuch as saccharin sodium, a volatile oil, a buffering agent, a surfaceactive agent, and/or a preservative such as methylhydroxybenzoate. Thedroplets provided by this route of administration may have an averagediameter in the range from about 0.1 to about 200 nanometers.

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition describedherein. Another formulation suitable for intranasal administration is acoarse powder comprising the active ingredient and having an averageparticle from about 0.2 to 500 micrometers. Such a formulation isadministered by rapid inhalation through the nasal passage from acontainer of the powder held close to the nares.

Formulations for nasal administration may, for example, comprise fromabout as little as 0.1% (w/w) to as much as 100% (w/w) of the activeingredient, and may comprise one or more of the additional ingredientsdescribed herein. A pharmaceutical composition described herein can beprepared, packaged, and/or sold in a formulation for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and maycontain, for example, 0.1 to 20% (w/w) active ingredient, the balancecomprising an orally dissolvable and/or degradable composition and,optionally, one or more of the additional ingredients described herein.Alternately, formulations for buccal administration may comprise apowder and/or an aerosolized and/or atomized solution and/or suspensioncomprising the active ingredient. Such powdered, aerosolized, and/oraerosolized formulations, when dispersed, may have an average particleand/or droplet size in the range from about 0.1 to about 200 nanometers,and may further comprise one or more of the additional ingredientsdescribed herein.

A pharmaceutical composition described herein can be prepared, packaged,and/or sold in a formulation for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1-1.0% (w/w) solution and/or suspension of the activeingredient in an aqueous or oily liquid carrier or excipient. Such dropsmay further comprise buffering agents, salts, and/or one or more otherof the additional ingredients described herein. Otheropthalmically-administrable formulations which are useful include thosewhich comprise the active ingredient in microcrystalline form and/or ina liposomal preparation. Ear drops and/or eye drops are alsocontemplated as being within the scope of this disclosure.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with ordinary experimentation.

Drugs provided herein can be formulated in dosage unit form for ease ofadministration and uniformity of dosage. It will be understood, however,that the total daily usage of the agents described herein will bedecided by a physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular subjector organism will depend upon a variety of factors including the diseasebeing treated and the severity of the disorder; the activity of thespecific active ingredient employed; the specific composition employed;the age, body weight, general health, sex, and diet of the subject; thetime of administration, route of administration, and rate of excretionof the specific active ingredient employed; the duration of thetreatment; drugs used in combination or coincidental with the specificactive ingredient employed; and like factors well known in the medicalarts.

The agents and compositions provided herein can be administered by anyroute, including enteral (e.g., oral), parenteral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,subcutaneous, intraventricular, transdermal, interdermal, rectal,intravaginal, intraperitoneal, topical (as by powders, ointments,creams, and/or drops), mucosal, nasal, bucal, sublingual; byintratracheal instillation, bronchial instillation, and/or inhalation;and/or as an oral spray, nasal spray, and/or aerosol. Specificallycontemplated routes are oral administration, intravenous administration(e.g., systemic intravenous injection), regional administration viablood and/or lymph supply, and/or direct administration to an affectedsite. In general, the most appropriate route of administration willdepend upon a variety of factors including the nature of the agent(e.g., its stability in the environment of the gastrointestinal tract),and/or the condition of the subject (e.g., whether the subject is ableto tolerate oral administration). In certain embodiments, the agent orpharmaceutical composition described herein is suitable for oraldelivery or intravenous injection to a subject.

The exact amount of an agent required to achieve an effective amountwill vary from subject to subject, depending, for example, on species,age, and general condition of a subject, severity of the side effects ordisorder, identity of the particular agent, mode of administration, andthe like. An effective amount may be included in a single dose (e.g.,single oral dose) or multiple doses (e.g., multiple oral doses). Incertain embodiments, when multiple doses are administered to a subjector applied to a tissue or cell, any two doses of the multiple dosesinclude different or substantially the same amounts of an agent (e.g., aTMED9-binding agent, e.g., BRD-4780) described herein.

As noted elsewhere herein, a drug of the instant disclosure may beadministered via a number of routes of administration, including but notlimited to: subcutaneous, intravenous, intrathecal, intramuscular,intranasal, oral, transepidermal, parenteral, by inhalation, orintracerebroventricular.

The term “injection” or “injectable” as used herein refers to a bolusinjection (administration of a discrete amount of an agent for raisingits concentration in a bodily fluid), slow bolus injection over severalminutes, or prolonged infusion, or several consecutiveinjections/infusions that are given at spaced apart intervals.

In some embodiments of the present disclosure, a formulation as hereindefined is administered to the subject by bolus administration.

A drug or other therapy of the instant disclosure is administered to thesubject in an amount sufficient to achieve a desired effect at a desiredsite (e.g., amelioration of kidney disease and/or symptoms, ameliorationof neurodegenerative disease and/or neurodegenerative disease-relatedsymptoms, reduction of retinitis pigmentosa symptoms, etc.) determinedby a skilled clinician to be effective. In some embodiments of thedisclosure, the agent is administered at least once a year. In otherembodiments of the disclosure, the agent is administered at least once aday. In other embodiments of the disclosure, the agent is administeredat least once a week. In some embodiments of the disclosure, the agentis administered at least once a month.

Additional exemplary doses for administration of an agent of thedisclosure to a subject include, but are not limited to, the following:1-20 mg/kg/day, 2-15 mg/kg/day, 5-12 mg/kg/day, 10 mg/kg/day, 1-500mg/kg/day, 2-250 mg/kg/day, 5-150 mg/kg/day, 20-125 mg/kg/day, 50-120mg/kg/day, 100 mg/kg/day, at least 10 μg/kg/day, at least 100 μg/kg/day,at least 250 μg/kg/day, at least 500 μg/kg/day, at least 1 mg/kg/day, atleast 2 mg/kg/day, at least 5 mg/kg/day, at least 10 mg/kg/day, at least20 mg/kg/day, at least 50 mg/kg/day, at least 75 mg/kg/day, at least 100mg/kg/day, at least 200 mg/kg/day, at least 500 mg/kg/day, at least 1g/kg/day, and a therapeutically effective dose that is less than 500mg/kg/day, less than 200 mg/kg/day, less than 100 mg/kg/day, less than50 mg/kg/day, less than 20 mg/kg/day, less than 10 mg/kg/day, less than5 mg/kg/day, less than 2 mg/kg/day, less than 1 mg/kg/day, less than 500μg/kg/day, and less than 500 μg/kg/day.

In certain embodiments, when multiple doses are administered to asubject or applied to a tissue or cell, the frequency of administeringthe multiple doses to the subject or applying the multiple doses to thetissue or cell is three doses a day, two doses a day, one dose a day,one dose every other day, one dose every third day, one dose every week,one dose every two weeks, one dose every three weeks, or one dose everyfour weeks. In certain embodiments, the frequency of administering themultiple doses to the subject or applying the multiple doses to thetissue or cell is one dose per day. In certain embodiments, thefrequency of administering the multiple doses to the subject or applyingthe multiple doses to the tissue or cell is two doses per day. Incertain embodiments, the frequency of administering the multiple dosesto the subject or applying the multiple doses to the tissue or cell isthree doses per day. In certain embodiments, when multiple doses areadministered to a subject or applied to a tissue or cell, the durationbetween the first dose and last dose of the multiple doses is one day,two days, four days, one week, two weeks, three weeks, one month, twomonths, three months, four months, six months, nine months, one year,two years, three years, four years, five years, seven years, ten years,fifteen years, twenty years, or the lifetime of the subject, tissue, orcell. In certain embodiments, the duration between the first dose andlast dose of the multiple doses is three months, six months, or oneyear. In certain embodiments, the duration between the first dose andlast dose of the multiple doses is the lifetime of the subject, tissue,or cell. In certain embodiments, a dose (e.g., a single dose, or anydose of multiple doses) described herein includes independently between0.1 μg and 1 μg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mgand 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g,inclusive, of an agent (e.g., a TMED9-binding agent, e.g., BRD-4780)described herein. In certain embodiments, a dose described hereinincludes independently between 1 mg and 3 mg, inclusive, of an agent(e.g., a TMED9-binding agent, e.g., BRD-4780) described herein. Incertain embodiments, a dose described herein includes independentlybetween 3 mg and 10 mg, inclusive, of an agent (e.g., a TMED9-bindingagent, e.g., BRD-4780) described herein. In certain embodiments, a dosedescribed herein includes independently between 10 mg and 30 mg,inclusive, of an agent (e.g., a TMED9-binding agent, e.g., BRD-4780)described herein. In certain embodiments, a dose described hereinincludes independently between 30 mg and 100 mg, inclusive, of an agent(e.g., a TMED9-binding agent, e.g., BRD-4780) described herein.

It will be appreciated that dose ranges as described herein provideguidance for the administration of provided pharmaceutical compositionsto an adult. The amount to be administered to, for example, a child oran adolescent can be determined by a medical practitioner or personskilled in the art and can be lower or the same as that administered toan adult. In certain embodiments, a dose described herein is a dose toan adult human whose body weight is 70 kg.

It will be also appreciated that an agent (e.g., a TMED9-binding agent,e.g., BRD-4780) or composition, as described herein, can be administeredin combination with one or more additional pharmaceutical agents (e.g.,therapeutically and/or prophylactically active agents), which aredifferent from the agent or composition and may be useful as, e.g.,combination therapies.

The agents or compositions can be administered in combination withadditional pharmaceutical agents that improve their activity (e.g.,activity (e.g., potency and/or efficacy) in treating a disease (e.g., atoxic proteinopathy resulting from mutant protein accumulation in theearly secretory pathway, such as a neurodegenerative disease, MKD, anautosomal dominant kidney disease caused by uromodulin mutation, a formof retinitis pigmentosa caused by rhodopsin mutation, etc.) in a subjectin need thereof, in preventing a disease in a subject in need thereof,in reducing the risk of developing a disease in a subject in needthereof, in inhibiting the replication of a virus, in killing a virus,etc. in a subject or cell. In certain embodiments, a pharmaceuticalcomposition described herein including an agent (e.g., a TMED9-bindingagent, e.g., BRD-4780) described herein and an additional pharmaceuticalagent shows a synergistic effect that is absent in a pharmaceuticalcomposition including one of the agent and the additional pharmaceuticalagent, but not both.

In some embodiments of the disclosure, a therapeutic agent distinct froma first therapeutic agent of the disclosure is administered prior to, incombination with, at the same time, or after administration of the agentof the disclosure. In some embodiments, the second therapeutic agent isselected from the group consisting of a chemotherapeutic, animmunotherapy (e.g., an agent for immune checkpoint blockade such as aPD-1 inhibitor, optionally with or without one or more CTLA-4inhibitors), an antioxidant, an antiinflammatory agent, anantimicrobial, a steroid, etc.

The agent or composition can be administered concurrently with, priorto, or subsequent to one or more additional pharmaceutical agents, whichmay be useful as, e.g., combination therapies. Pharmaceutical agentsinclude therapeutically active agents. Pharmaceutical agents alsoinclude prophylactically active agents. Pharmaceutical agents includesmall organic molecules such as drug compounds (e.g., compounds approvedfor human or veterinary use by the U.S. Food and Drug Administration asprovided in the Code of Federal Regulations (CFR)), peptides, proteins,carbohydrates, monosaccharides, oligosaccharides, polysaccharides,nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides orproteins, small molecules linked to proteins, glycoproteins, steroids,nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides,antisense oligonucleotides, lipids, hormones, vitamins, and cells. Incertain embodiments, the additional pharmaceutical agent is apharmaceutical agent useful for treating and/or preventing a diseasedescribed herein. Each additional pharmaceutical agent may beadministered at a dose and/or on a time schedule determined for thatpharmaceutical agent. The additional pharmaceutical agents may also beadministered together with each other and/or with the agent orcomposition described herein in a single dose or administered separatelyin different doses. The particular combination to employ in a regimenwill take into account compatibility of the agent described herein withthe additional pharmaceutical agent(s) and/or the desired therapeuticand/or prophylactic effect to be achieved. In general, it is expectedthat the additional pharmaceutical agent(s) in combination be utilizedat levels that do not exceed the levels at which they are utilizedindividually. In some embodiments, the levels utilized in combinationwill be lower than those utilized individually.

The additional pharmaceutical agents include, but are not limited to,other kidney disease, neurodegenerative disease and/or retinitispigmentosa therapies, anti-cancer agents, immunotherapy and/orimmunomodulatory agents, anti-proliferative agents, cytotoxic agents,anti-angiogenesis agents, anti-inflammatory agents, immunosuppressants,anti-bacterial agents, anti-viral agents, cardiovascular agents,cholesterol-lowering agents, anti-diabetic agents, anti-allergic agents,contraceptive agents, and pain-relieving agents. In certain embodiments,the additional pharmaceutical agent is an anti-viral agent. In certainembodiments, the additional pharmaceutical agent is selected from thegroup consisting of epigenetic or transcriptional modulators (e.g., DNAmethyltransferase inhibitors, histone deacetylase inhibitors (HDACinhibitors), lysine methyltransferase inhibitors), antimitotic drugs(e.g., taxanes and vinca alkaloids), hormone receptor modulators (e.g.,estrogen receptor modulators and androgen receptor modulators), cellsignaling pathway inhibitors (e.g., tyrosine kinase inhibitors),modulators of protein stability (e.g., proteasome inhibitors), Hsp90inhibitors, glucocorticoids, all-trans retinoic acids, and other agentsthat promote differentiation. In certain embodiments, the agentsdescribed herein or pharmaceutical compositions can be administered incombination with a kidney, eye or other therapy including, but notlimited to, dialysis, surgery, transplantation (e.g., kidneytransplantation, stem cell transplantation, etc.), immunotherapy, and/orchemotherapy.

Dosages for a particular agent of the instant disclosure may bedetermined empirically in individuals who have been given one or moreadministrations of the agent.

Administration of an agent of the present disclosure can be continuousor intermittent, depending, for example, on the recipient'sphysiological condition, whether the purpose of the administration istherapeutic or prophylactic, and other factors known to skilledpractitioners. The administration of an agent may be essentiallycontinuous over a preselected period of time or may be in a series ofspaced doses.

Guidance regarding particular dosages and methods of delivery isprovided in the literature; see, for example, U.S. Pat. No. 4,657,760;5,206,344; or 5,225,212. It is within the scope of the instantdisclosure that different formulations will be effective for differenttreatments and different disorders, and that administration intended totreat a specific organ or tissue may necessitate delivery in a mannerdifferent from that to another organ or tissue. Moreover, dosages may beadministered by one or more separate administrations, or by continuousinfusion. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until a desiredsuppression of disease symptoms occurs. However, other dosage regimensmay be useful. The progress of this therapy is easily monitored byconventional techniques and assays.

Kits

The instant disclosure also provides kits containing agents of thisdisclosure for use in the methods of the present disclosure. Kits of theinstant disclosure may include one or more containers comprising anagent (e.g., a TMED9-binding agent, e.g., BRD-4780) of this disclosureand/or may contain agents (e.g., oligonucleotide primers, probes, etc.)for identifying a kidney disease, eye disease and/or neurodegenerativedisease associated with a toxic proteinopathy resulting from mutantprotein accumulation in the early secretory pathway in a subject, cell,tissue and/or organoid. In some embodiments, the kits further includeinstructions for use in accordance with the methods of this disclosure.In some embodiments, these instructions comprise a description ofadministration of the agent to treat or diagnose, e.g., a toxicproteinopathy resulting from mutant protein accumulation in the earlysecretory pathway, according to any of the methods of this disclosure.In some embodiments, the instructions comprise a description of how todetect a toxic proteinopathy resulting from mutant protein accumulationin the early secretory pathway, for example in an individual, in atissue sample, or in a cell. The kit may further comprise a descriptionof selecting an individual suitable for treatment based on identifyingwhether that subject has a toxic proteinopathy resulting from mutantprotein accumulation in the early secretory pathway and/or a biomarker(e.g., a MUC1-fs, C126R UMOD and/or P23H rhodopsin mutant) indicative ofa toxic proteinopathy resulting from mutant protein accumulation in theearly secretory pathway.

The instructions generally include information as to dosage, dosingschedule, and route of administration for the intended treatment. Thecontainers may be unit doses, bulk packages (e.g., multi-dose packages)or sub-unit doses. Instructions supplied in the kits of the instantdisclosure are typically written instructions on a label or packageinsert (e.g., a paper sheet included in the kit), but machine-readableinstructions (e.g., instructions carried on a magnetic or opticalstorage disk) are also acceptable.

The label or package insert indicates that the composition is used fortreating, e.g., a toxic proteinopathy resulting from mutant proteinaccumulation in the early secretory pathway, in a subject. Instructionsmay be provided for practicing any of the methods described herein.

The kits of this disclosure are in suitable packaging. Suitablepackaging includes, but is not limited to, vials, bottles, jars,flexible packaging (e.g., sealed Mylar or plastic bags), and the like.Also contemplated are packages for use in combination with a specificdevice, such as an inhaler, nasal administration device (e.g., anatomizer) or an infusion device such as a minipump. A kit may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The container may also have a sterile access port(e.g., the container may be an intravenous solution bag or a vial havinga stopper pierceable by a hypodermic injection needle). In certainembodiments, at least one active agent in the composition is aTMED9-binding agent, e.g., BRD-4780. The container may further comprisea second pharmaceutically active agent.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container.

The practice of the present disclosure employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989.Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rdEd. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.);Ausubel et al., 1992), Current Protocols in Molecular Biology (JohnWiley & Sons, including periodic updates); Glover, 1985, DNA Cloning(IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow andLane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Cabs eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6thEdition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. Aguide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ.of Oregon Press, Eugene, 2000).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present disclosure, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Reference will now be made in detail to exemplary embodiments of thedisclosure. While the disclosure will be described in conjunction withthe exemplary embodiments, it will be understood that it is not intendedto limit the disclosure to those embodiments. To the contrary, it isintended to cover alternatives, modifications, and equivalents as may beincluded within the spirit and scope of the disclosure as defined by theappended claims. Standard techniques well known in the art or thetechniques specifically described below were utilized.

EXAMPLES Example 1: Materials and Methods

Human Kidney Biopsies

Kidney tissue was obtained as discarded tissue from surgicalnephrectomies performed for clinical indications. Control samples wereexamined by a renal pathologist to select tissue for further processing.For MKD patient tissue, samples from kidney cortex and medulla werecollected from a 50 year old female patient with advanced diseaseundergoing nephrectomy for a suspicious cyst that was proved benign.

hMUC1 knock-in replacement mice wt/+ and fs/+ knock-in (KI) 129S2 micewere generated by GenOway (Lyon, France) using embryonic stem (ES) cellsgenetically modified by homologous recombination, through KI of theentire human MUC1 gene into the murine MUC1 locus. Mice were maintainedon a 12 hr light/dark cycle at 18-26° C. in an AAALAC accreditedfacility and fed ad libitum with water and PicoLab® Rodent Diet 20pellets (0007688, LabDiet). All animal experiments were approved by theInstitutional Animal Care and Use Committee (IACUC) at The BroadInstitute of MIT and Harvard and were conducted in accordance withNational Institutes of Health (NIH) animal research guidelines.

Normal and MKD Patient Derived iPS Cell Lines

iPS cell lines from three MKD patients and their unaffected siblingswere derived from erythroblasts using CTS™ CytoTune™-iPS 2.1 SendaiReprogramming Kit (A34546, Thermo Fisher Scientific®) at the HarvardStem Cell Institute iPS Core Facility. Cell lines were characterized forpluripotency and spontaneous differentiation to the three germ layersusing qPCR based on standard protocols at the HSCI Core Facility. AlliPSC cultures were maintained in mTeSR1 medium in T25 flasks coated withMatrigel. Cells were passaged using Gentle Cell Dissociation Reagent.All lines were confirmed to be karyotype normal and maintained belowpassage 15. Cell lines were routinely checked and were negative formycoplasma.

HEK293T Cells

HEK293T cells (ATCC) were cultured according to standard protocol inDulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FetalCalf Serum (26140079, Life Technologies), 100 units/mL penicillin and100 μg/mL streptomycin (15140-163, Life Technologies).

Kidney Epithelial Cells

N and P cells, generated herein, were cultured in Renal Life BasalMedium (102970-948, VWR) supplemented with Renal Life Factors(102970-862, VWR), with the exclusion of Gentamycin and Amphotericin B.For all experiments, P cells were maintained below passage 12.

AtT-20 Pituitary Cells Expressing UMOD

AtT-20 mouse pituitary cells (ATCC® CCL-89™) stably transfected withcDNAs encoding WT-UMOD or UMOD mutant C126R were maintained inDMEM:Nutrient Mixture F-12 (DMEM/F12) (10565042, Thermo FisherScientific®) supplemented with 10% Fetal Calf Serum (26140079, LifeTechnologies), 100 units/mL penicillin and 100 μg/mL streptomycin(15140-163, Life Technologies) and 0.8 mg/mL of G418 (04727878001,Sigma-Aldrich).

MUC1-wt and MUC1-fs Plasmid Design

Plasmid constructs for in vivo expression of MUC1-wt and MUC1-fsproteins were based on the normal and mutant MUC1 alleles of MKD patientF6:IV-3, each containing a VNTR region comprising 37 near-identical60-mer repeat units that had been completely sequenced and assembledpreviously (Kirby et al., 2013). The frameshift mutation is found in thesecond repeat unit of this allele. The MUC1 tandem array and flankingregions were PCR-amplified in 50 μl containing 50 ng genomic DNA, 15pmol forward (GGCAGAGAAAGGAAATGGCACATCACT; SEQ ID NO: 6) and 15 pmolreverse primer (CTGCTGCTCCTCACAGTGCTTACAGGT; SEQ ID NO: 7), 0.2 mM ofeach dNTP and 1.25 U PrimeSTAR GXL DNA Polymerase in 1× PrimeSTAR GXLbuffer (Takara). The thermoprofile was 1 min at 94° C., 22 cycles of 10sec at 98° C., 10 sec at 65° C., 6 min at 68° C., with final extensionfor 10 min at 68° C. Bona fide full-length PCR products spanningchr1:155,160,024-155,162,601 of the incomplete hg19 reference genome(4.15 kb in F6:IV-3) were gel-purified, cleaned up by QiaQuickgel-extraction kits (Qiagen) and TOPO-TA cloned in pCR-4-TOPO vector inTOP10 cells (Thermo Fisher Scientific®). Transformants were Sangersequenced with vector and insert primers to determine the frameshiftmutation status and to screen out clones harboring PCR inducednon-synonymous mutations within reach of unique sequencing primers, i.e.flanking the 1.3 kb inaccessible core of the tandem repeat. Clonespassing this test were then subjected to full-length Pacific Biosciencessequencing of a 4.7 kb fragment generated by digestion with NspI andPvuI. The clone chosen for MUC1-fs expression constructs included asynonymous PCR-induced mutation of a leucine codon located 731 codonsdownstream of the codon altered by the frameshift mutation.

Construction of MUC1-wt and MUC1-fs Plasmids for Generation of Knock-inMice

To replace one copy of the endogenous mouse Muc1 gene with WT and mutantalleles of the human MUC1 gene, including its regulatory sequences (FIG.1A), the 3.9 kb DraIII fragment was subcloned into an acceptor vectorproviding sequences upstream and downstream sequences of hg19chr1:155157737-155165183 which contains the MUC1 gene body, the promoterand enhancer region up to and including micro RNA gene MIR92B (FIG. 7A),as well as flanking restriction sites required for the generation andanalysis of KI transgenic mice generated by GenOway (Lyon, France).Before assembling the constructs, two synonymous single-base changeswere introduced to the normal MUC1 sequence. Both artificial markersdisrupt a restriction site and thus provide a simple genotyping assayfor tracking and identification purposes. Marker 1 is a G to Asubstitution at hg19 chr1:155,161,995, which disrupts a BseY1 site in aPCR product generated with primers GCTACCACAGCCCCTAAACC (SEQ ID NO: 8)and GCTGTGGCTGGAGAGTACG (SEQ ID NO: 9). Marker 2 is a T to Asubstitution at hg19 chr1: 155,160,802, which disrupts a KpnI site in aPCR product generated with primers CCAGCCATAGCACCAAGACT (SEQ ID NO: 10)and GGAAGGAAAGGCCGATACTC (SEQ ID NO: 11). The modified VNTR-containingWT clone was verified by full-length Pacific Biosciences sequencing asbefore. MUC1 transgene fragments were assembled in the BigEasy v2.0linear cloning vector pJazz-OK (Lucigen), which reduces torsional stressduring replication and helps maintain otherwise unstable anddifficult-to-clone sequences (Godiska et al., 2010). The finalconstructs underwent three independent sequence-validation checks: i)Illumina® Mi-Seq sequencing with paired-end 150 base reads and pilonanalysis (Walker et al., 2014) that detected errors in the vectorsequence and none in the cloned insert but has blind spots in the tandemrepeat region, ii) full-length Pacific Biosciences sequencing of the3.9-kb DraIII fragment excised from the linear pJazz clones, and iii)subcloning of the 3.9-kb DraIII in a low-copy number pSMART LC-Kanvector (Lucigen), followed by transposon hopping for deep bidirectionalSanger sequencing and tandem-repeat assembly. This sequence validationemployed the same approach that was used for the original identificationof the frameshift mutation in MKD patients (Kirby et al., 2013). Thefinal constructs (22 kb including the vector) were provided to GenOwayfor KI transgenesis and development of a mouse model for MKD.

UMOD Plasmid

Wild-type UMOD mRNA was reverse-transcribed from human total kidney RNA,PCR amplified, and cloned into a pCR3.1 vector (Thermo FisherScientific®, Thermo Fisher Scientific®, California, USA). Constructswere introduced into E. coli TOP 10′F strain (Thermo Fisher Scientific®,California, USA), and candidate recombinant clones were confirmed bysequencing. Mutant UMOD construct c.385T>C (C126R-UMOD) was prepared bysite-directed mutagenesis of the WT-UMOD/pCR3.1 construct.

Rhodopsin Plasmid

P23H mutant-GFP rhodopsin plasmid in pEGFP-N1 vector was kindly providedby Michael E. Cheetham.

Huntingtin Plasmid

Plasmid of huntingtin gene exon 1 fragments containing 97 CAG (97Q)repeats fused to GFP was kindly provided by Steven Finkbeiner.

Isolation and Immortalization of Kidney Epithelial Cells

Kidney tissues from control and MKD patient were finely minced, digestedwith 1 mg/mL Collagenase-type II (Worthington Corp) at 37° C. for 30 minand passed through a series of sieves (100 μm, 70 μm). Tubules that wereretained on the top of 70 μm sieve were plated in RenalLife Epithelialmedium on collagen coated plates for propagation. Next, cells wereimmortalized using lentivirus carrying human Telomerase ReverseTranscritpase (hTERT) produced in HEK293T cells. Briefly, viralsupernatant was added to cells in the presence of 1 μg/mL Polybrene.Cells were then spun at 2000 rpm for 1 hr at 30° C., then washed withDMEM/F12 (10565042, Thermo Fisher Scientific®) and incubated in RenaLifeEpithelial Medium for 24 hr, followed by a second identical cycle ofviral transduction. Transduced cells were selected and expanded in 100μg/mL of Hygromycin B (10687010, Thermo Fisher Scientific®), then clonedby serial dilution. Clones of control (N) cells and MKD patient (P)cells were selected for MUC1 protein abundance and cell polarization onCorning Transwell® Semipermeable support plates (07-200-560, ThermoFisher Scientific®).

Generation of Kidney Organoids

Kidney organoids were generated as previously described (Takasato etal., 2016) with slight modifications. The iPS cells generated from MKDpatients and their unaffected siblings were seeded at 375,000 cells perT25 flask in mTeSR1 media (85870, Stem Cell Technologies) supplementedwith 10 μM/mL Rock Inhibitor Y-27632 (72304, Stem Cell technologies).After 24 hr, cells were treated with 8 μM CHIR 99021 (4423/10, R&Dsystems) in STEMdiff™ APEL™2 Medium (05270, Stem cell Technologies) for4 days, followed by addition of 200 ng/mL Recombinant Human FGF-9(100-23, Peprotech) and 1 μg/mL heparin (H4784, Sigma-Aldrich) for 3more days. At day 7, cells were dissociated into single cells usingACCUTASE™ (07920, Stem Cell Technologies) at 37° C. for 5 min. Then,500,000 cells were pelleted at 350×g for 2 min (twice with 180° flipafter first spin) and transferred onto a transwell membrane (3450,Corning). Pellets were incubated with 5 μM CHIR 99021 in STEMdiff™APEL™2 Medium for 1 hr. Afterwards, 200 ng/mL Recombinant Human FGF-9and heparin were added for 5 days, followed by 2 days incubation with 1μg/mL heparin. For the following 15 days, organoids were kept inSTEMdiff™ APEL™2 Medium. Media were changed every other day.

MUC1 KI Replacement Mice

The wt/+ and fs/+ KI replacement mice were developed by GenOway (Lyon,France) using human MUC1-wt and MUC1-fs constructs described above. TheMUC1 constructs were knocked into ES cells using homologousrecombination. ES cells were injected into blastocysts, and chimeric129S2 mice were generated. Following breeding, heterozygous miceexpressing either hMUC1-wt or hMUC1-fs were obtained.

Genotyping

Mouse genotyping was performed by Transnetyx, using real-time PCR toidentify mice expressing the human MUC1 versus the mouse MUC1 version.

Pharmacokinetics Studies

1) A pharmacokinetic study was performed by WuXi AppTec (Hong Kong) infasted male 12952/SvPasCrl mice (study #400565-20170331-1V1PK) todetermine the oral bioavailability of BRD-4780. A single dose ofBRD-4780 was administered at 3.0 mg/kg intravenously (i.v.) or 10.0mg/kg orally (p.o) in a clear solution of 10% HP-β-CD in saline. Bloodwas collected serially from n=3 mice per dose group at eight time pointspost dose administration (0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours) andplasma was obtained by centrifugation. Plasma drug concentration wasdetermined by LC-MS/MS and reported as BRD-4780 ng/mL for plasma andng/mg for kidney tissue, and plotted as the mean±standard deviationconcentration (FIG. 11A). Standard pharmacokinetic parameters werecalculated (FIG. 11B). The oral bioavailability (% F) was determined tobe 119%. In plasma protein binding studies, the estimated percentprotein bound was 29.7%.

2) A pharmacokinetic study for BRD-4780 was performed by WuXi AppTec(Hong Kong) in fed 12952/SvPasCrl male (study 400565-2018051001-MPK) andfemale mice. A single dose of BRD-4780 was administered orally at 10.0mg/kg, 20 mg/kg, or 50 mg/kg in a clear solution of 10% HP-β-CD insaline. “BRD-8507” was also listed in this study; BRD-8507 is the samecompound as BRD-4780 but it is an alternative batch of the compound.Blood and kidney tissue were collected from n=3 mice per dose group ateight time points post dose administration (0.25, 0.5, 1, 2, 4, 6, 8 and24 hours). Plasma was obtained by centrifugation and the kidney tissuewas homogenized. Plasma and tissue drug concentrations were determinedby LC-MS/MS and reported as BRD-4780 ng/mL for plasma and ng/mg forkidney tissue, and plotted as the mean f standard deviationconcentration for males (FIG. 11C) and females (FIG. 11D). Standardpharmacokinetic parameters were calculated for males (FIG. 11E) andfemales (FIG. 11F).

3) A pharmacokinetic study was performed by Charles River Labs (CRL)(Worcester, Mass., USA) in fed male 129-ELITE mice (study EF-0022-DA-MI)to compare the oral bioavailability of BRD-1365 and BRD-7709 toBRD-4780. BRD-1365 has the following structure:

BRD-7709 has the structure:

A single dose of BRD-4780, BRD-1365 or BRD-7709 was administered at 1.0mg/kg intravenously (i.v.) or 5.0 mg/kg orally (p.o) in a clear solutionof 5% dextrose in water (D5W). Blood was collected serially from n=3mice per dose group at seven time points post i.v. dose administration(0.083, 0.25, 0.5, 1, 2, 4 and 24 hours) or six time points post p.o.dose administration (0.25, 0.5, 1, 2, 4 and 24 hours) and plasmaobtained by centrifugation. Plasma drug concentration was determined byLC-MS/MS and reported as ng/mL in plasma plotted as the mean±standarddeviation concentration for BRD-4780 (FIG. 11G), BRD-1365 (FIG. 11H) andBRD-7709 (FIG. 11I). Standard pharmacokinetic parameters were calculated(FIG. 11J).

4) A pharmacokinetic study was performed by Charles River Labs (CRL)(Worcester, Mass., USA) in fed male 129-ELITE mice (study EF-0024-DA-MI)to determine the dose response exposure of BRD-4780, BRD-1365 andBRD-7709 in plasma, kidney, liver and eye. Brain exposure to BRD-4780 at30 mg/kg was also evaluated A single dose of BRD-4780 was administeredat 30.0 mg/kg or 100 mg/kg orally (p.o) in a clear solution of 5%dextrose in water (D5W). For BRD-1365 and BRD-7709, a single dose ofeither compound was administered at 10.0 mg/kg, 30.0 mg/kg, 50.0 mg/kgor 100 mg/kg orally (p.o) in a clear solution of 5% dextrose in water(D5W). Blood, kidney, liver and eye tissue were collected from n=3 miceper dose group at six time points post p.o. dose administration (1, 2,6, 10, 16 and 24 hours) with all three compounds. Brain tissue wascollected from n=3 mice per dose group at six time points post p.o. doseadministration (1, 2, 6, 10, 16 and 24 hours) with BRD-4780 at 30 mg/kg.Plasma obtained by centrifugation and tissue fragments were homogenized.Plasma and tissue drug concentration was determined by LC-MS/MS andreported as drug concentration in ng/mL for plasma and ng/mg for kidney,liver and eye tissue, plotted as the mean±standard deviationconcentration for males for BRD-4780 (FIG. 11K), BRD-1365 (FIG. 11K) andBRD-7709 (FIG. 11M). Standard pharmacokinetic parameters were calculatedfor males for BRD-4780 (FIG. 11B), BRD-1365 (FIG. 11O) and BRD-7709(FIG. 11P). All three compounds had a high volume of distribution, withhighest exposures in the eye. C_(max) exposures for doses above 10 mg/kgwere not dose proportional. AUC exposures for BRD-4780 and BRD-1365 werenot dose proportional, but BRD-7709 AUC exposures were nearly doseproportional.

5) A pharmacokinetic study was performed by WuXi AppTec (NJ, USA) in fedmale Sprague Dawley rats (study BIMH-20200211-RPK) to determine the oralbioavailability of BRD-4780, BRD-7709 and BRD-1365 in rats. A singledose of BRD-4780, BRD-1365 or BRD-7709 was administered at 1.0 mg/kgintravenously (i.v.) or 5.0 mg/kg orally (p.o) in a clear solution of 5%dextrose in water (D5W). Blood was collected serially from n=2 rats perdose group at nine time points post i.v. dose administration (0.083,0.5, 1, 3, 6, 10, 24, 32 and 48 hours) or nine time points post p.o.dose administration (0.25, 0.5, 1, 3, 6, 10, 24, 32 and 48 hours) andplasma obtained by centrifugation. Plasma drug concentration wasdetermined by LC-MS/MS and reported as ng/mL in plasma. Plasmaconcentrations vs. time for individual rats and the mean of 2 rats pergroup were plotted for BRD-4780 (FIG. 11Q), BRD-1365 (FIG. 11R) andBRD-7709 (FIG. 11S). Standard pharmacokinetic parameters were calculated(FIG. 11T). The oral bioavailability (% F) was determined to be 40.6%.for BRD-4780, 0.083, 0.5, 1, 3, 6, 10, 24, 32 and 48 hours 32.7% forBRD-1365 and 56.1% for BRD-7709), with BRD-7709 showing the highest oralbioavailability, plotted in FIG. 11U.

6) A pharmacokinetic study was performed by WuXi AppTec (Wuhan, China)in fed male and female CD(SD) rats (study BIMH-2020042701-RPK(BRD-4780), BIMH-2020042702-RPK (BRD-7709), and BIMH-2020042703-RPK(BRD-1365) inclusive) to determine the dose response exposure ofBRD-4780, BRD-7709 and BRD-1365 following single oral administrations inmale and female CD(SD) rats. A single dose of BRD-4780, BRD-1365 orBRD-7709 was orally administered at 10.0 mg/kg (p.o.), 30.0 mg/kg(p.o.), 50.0 mg/kg (p.o.) or 100.0 mg/kg (p.o.) in a clear solution of5% dextrose in water (D5W). Blood was collected serially from n=3 ratsper dose group, at nine time points post dose administration (0.25, 0.5,1, 3, 6, 10, 16, 24, and 48 hours). Plasma was obtained bycentrifugation. Plasma drug concentration was determined by LC-MS/MS andreported as ng/mL in plasma. Plasma concentrations vs. time forindividual rats and the mean of 3 rats per group were plotted forBRD-4780 in male rats (FIG. 11V) and female rats (FIG. 11W). Plasmaconcentrations vs. time for individual rats and the mean of 3 rats pergroup were plotted for BRD-7709 in male rats (FIG. 11X) and female rats(FIG. 11Y). Plasma concentrations vs. time for individual rats and themean of 3 rats per group were plotted for BRD-1365 in male rats (FIG.11Z) and female rats (FIG. 11AA). The 24 hour and 48 hour time pointsfor one of three male rats in the BRD-1365 10 mg/kg group were excludedas likely technical outliers. Standard pharmacokinetic parameters werecalculated for BRD-4780 (FIG. 11AB), BRD-7709 (FIG. 11AC) and BRD-1365(FIG. 11AD). In contrast to mice, in which AUC exposure values werehigher in male mice than in female mice, AUC values were 1.4 to 2.3 foldhigher in female rats than in male rats.

Plasma Collection and Creatinine Quantification

Monthly blood samples from conscious mice were collected into lithiumheparin with plasma separator tubes (365965, BD Microtainer) by 4 mmlancet puncture of the submandibular vein (504540, World PrecisionInstruments), alternating right and left sides. Samples were centrifugedat 2000×g at 4° C. for 10 min, with transfer of plasma to an Eppendorf™DNA LoBind Microcentrifuge Tube (022431021), followed by anothercentrifugation at 2000×g 2000 for 10 min at 4° C. Plasma samples werekept at −80° C. until sent for creatinine measurement to UAB BiochemicalGenetics Laboratory, University of Alabama.

Mouse Transcardial Perfusion

Transcardial perfusion-fixation was performed for testing MUC1-fscolocalization with TMED9-positive vesicles (FIG. 5C). Mice wereanesthetized with 3 L/min of 3% isoflurane in 02 for 5 min (Combi-vet®system; Rothacher Medical, Bern, Switzerland). Anesthetized mice weretranscardially perfused with 0.1 M PBS (pH 7.4) followed by 4% PFA in0.1 M PBS. Kidneys were removed, transected into halves and postfixed in4% PFA for 2 hr at 4° C. Fixed tissue was rinsed in 0.1 M PBS, thencryoprotected 1 hr with 10% sucrose, 5 hr with 20% sucrose, andovernight in 30% sucrose. Cryoprotected tissue was mounted in OCT in adry ice-ethanol bath and processed for IF studies.

Lentivirus Production

Lentiviral stocks were generated by transfection of HEK293T cells. Cellswere transfected with the lentiviral target vector together with a2^(nd) generation packaging plasmid containing gag, pol and rev genes(e.g. pCMV-dR8.91), and a VSV-G expressing plasmid using TranslT-LT1transfection reagent (Mirus Bio, MIR 2300/5/6) as recommended by themanufacturer. Lentiviral supernatants were collected at 48 hr and 72 hrpost transfection, then were passed through a 0.45 mm filter and applieddirectly to cells or aliquoted and frozen.

RNA Interference

For knockdown of nischarin (putative I1R)(FIG. 14C), a short hairpin RNA(shRNA) plasmid construct (TRCN0000256843) was used to generatelentiviral stocks. For empty vector (EV) control, a similar plasmidlacking the nischarin shRNA was used (TRCN0000208001). P cells weretransduced with either targeting shRNA or EV.

CRISPR Cas9 System

To generate Cas9-expressing P cells, the Cas9 expression vectorpXPR_BRD111 was used. For knockout of nischarin (FIG. 14C), lentiviralstocks expressing sgRNA plasmid constructs (BRDN0001486234;KO1,BRDN0001482682;KO2) were used. For TMED9 knockout (FIG. 6B), lentiviralstocks expressing sgRNA plasmid constructs (BRDN0003481199;KO1,BRDN0003481863;KO2) were used. Non-targeting sgRNAs(BRDN0001148129;NTC1, BRDN0001146004;NTC2) were used as controls.Cas9-expressing P cells were assayed for Cas9 activity using pXPR_BRD047plasmid which expresses eGFP and an sgRNA targeting eGFP.

Lentivirus Transduction

P cells were transduced with either lentiviral stocks of nischarin shRNAor Cas9 expressing vector. Viral supernatants were applied to cells for24 hr in the presence of 4 μg/mL protamine sulfate (194729MP,Biomedicals). Infected cells were washed three times to remove viralparticles and transduced cells were selected in either 2 μg/mL puromycin(A11138-02, Thermo Fisher Scientific®)(for nischarin shRNA transduction)or 8 μg/mL Blasticidin (A11139-03, Thermo Fisher Scientific®)(for stableCas9 expression). For sgRNA transduction, Cas9-expressing P cells weretransduced as described above and selection was made using 2 μg/mLpuromycin.

Transfection of Rhodopsin Constructs

N cells plated in 96-well plates were transfected with 20 ng/well ofP23H mutant rhodopsin-GFP plasmid DNA using Lipofectamine 3000 (ThermoFisher Scientific®). Eight hr post-transfection, the cells were washedand processed as described below.

Stable Transfection of UMOD Constructs

AtT20 cells at 75% confluence were transfected with 4 μg DNA usingLipofectamine 2000 (Thermo Fisher Scientific®). After 72 hr, cells weretrypsinized, diluted, and selected in 0.8 mg/mL G418 (Thermo FisherScientific®). UMOD-expressing clones were selected for further studyusing PCR, DNA sequencing, and western blot analyses.

Stable Transfection of Huntingtin Constructs

HEK293T cells were transfected with Huntingtin 97Q repeats fused to GFPand selected in 0.8 mg/mL G418 (Thermo Fisher Scientific®).

Live Fluorescence Imaging of Cell Death and Apoptosis

To study apoptosis and cell death in N and P cells, 384 well CellCarrier Ultra plates (6057308, Perkin Elmer), pre-coated with 0.25 mg/mLSynthemax II SC Substrate (3535, Corning) were used. For UPR branchactivation experiments (FIGS. 2B, 9I, 9J and 9G) N and P cells weretreated as described in the corresponding figure legends. CellEventCaspase-3/7 Green Detection Reagent (C10423, Thermo Fisher Scientific®)and DRAQ7 (DR71000, Biostatus) were used at 1:5000 to monitor apoptosisand cell death respectively. Cells were imaged daily during 4 days tomonitor viability and image analysis was performed as described below.For rescue experiments, P cells were plated on 96 well Cell CarrierUltra microplates (6055302, Perkin Elmer) at 30,000 cells/well andcultured for 24 hr. BRD-4780 (5 μM) was applied as a pretreatment for 48hr prior to thapsigargin exposure and throughout the experiment.Thapsigargin was applied at increasing doses and the plates were imageddaily thereafter. To monitor cell death in N cells transientlyexpressing P23H mutant rhodopsin, DRAQ7 (1:5000) was added with DMSO or5 μM BRD-4780 treatments. Treated plates were imaged 30 min post-dyeapplication and daily thereafter.

Cell Immunofluorescence (IF) Staining

Cells grown on CellCarrier-96 or -384 well Ultra microplates(PerkinElmer) were fixed 10 min in PBS containing 4% PFA (ElectronMicroscopy Sciences), permeabilized 15 min in 0.5% Triton X100(X100-100ML, Sigma-Aldrich), blocked for 1 hr in blocking reagent (100mM Tris HCL pH8; 150 mM NaCL; 5 g/L Blocking Reagent [11096176001,Roche]) and treated for 1 hr with primary antibodies diluted in blockingreagent (1:500, monoclonal Fab-A-V5H anti-fsMUC1, AbD22655.2, Bio-Rad;1:2000, monoclonal Mouse anti-MUC1 (CD227), 60137, StemcellTechnologies; 1:200, polyclonal, Mouse anti-GM130, ab169276, Abcam;1:100, polyclonal, Rabbit anti-TMED9, 216201-AP, Proteintech; 1:800,polyclonal, Rabbit anti-SEC31A, 17913-1-AP, Proteintech; 1:400,monoclonal, Rabbit anti-EEA1 (C45B10), 3288S, Cell signaling technology;1:100, polyclonal, Rabbit anti-ERGIC-53, E1031, Sigma-Aldrich; 1:400,monoclonal, Mouse anti-LAMP1 (D4O1S), 15665, Cell signaling technology;1:1000, polyclonal, Rabbit anti-Calnexin, ab22595, Abcam; 1:2000,monoclonal, Rabbit anti-GM130 (D6B1) XP, 12480, Cell signalingtechnology; 1:200, monoclonal, Rabbit anti TNG46, ab50595, Abcam; 1:100,monoclonal, Rabbit anti-Rab7 (D95F2) XP, 9367, Cell signalingtechnology; 1:100, monoclonal, Rabbit anti-Rab11 (D4F5) XP, 5589, Cellsignaling technology). Fixed, stained cells were washed three times inPBS and incubated for 1 hr with secondary antibodies in blockingsolution (1:500, Alexa Fluor 488® Goat anti-Mouse IgG, A-11029, ThermoFisher Scientific®; 1:500, Alexa Fluor 647® F(ab′)2-Goat anti-RabbitIgG, A21246, Thermo Fisher Scientific®; 1:2000, Hoechst 33342, H3570,Thermo Fisher Scientific®), then washed three times in PBS and imaged.

Kidney Organoid IF Staining

Kidney organoids were immersion-fixed for 15 min in 4% PFA at 4° C. andsubsequently frozen in OCT using dry ice and 100% ethanol. Six pm-thickcryostat sections (CM1950, Leica) were thaw-mounted on microscope slides(Fisherbrand™ Superfrost™ Plus, Fisher Scientific) and kept in thecryostat (at −26° C.) for the duration of the sectioning process. Priorto immunostaining, organoid sections were rinsed in PBS for 5 min,incubated for 20 min at room temperature (RT) in PBS blocking solutioncontaining 5% normal donkey serum (S30-100ML, EMD Millipore) and 1.5%Tween® 20 (P1379, Sigma-Aldrich), then incubated overnight at 4° C. withprimary antibodies diluted in the same blocking solution (1:500,monoclonal Armenian hamster anti-MUC1, Ab80952, Abcam; 1:500, monoclonalFab-A-V5H anti-fsMUC1, AbD22655.4, Bio-Rad; 1:300 monoclonal Rat anti-ECadherin [DECMA-1], ab11512, Abcam; 1:500 polyclonal Rabbitanti-Laminin, L9393-100UL, Sigma-Aldrich; 1:500 Na/K-ATPase, ab76020,Abcam; 1:300 Fluorescein labeled Lotus Tetragonolobus Lectin (LTL),FL-1321, Vector laboratories; 1:100, polyclonal, Rabbit anti-TMED9,21620-1-AP, Proteintech). Immunostained organoid sections were thenrinsed three times for 10 min in PBS and incubated for 2 hr at RT withsecondary antibodies diluted in PBS containing 1.5% Tween-20 (1:500,Alexa Fluor® 488-conjugated AffiniPure F(ab′)2 Fragment Goat anti-HumanIgG, Jackson Immunoresearch; 1:500, Alexa Fluor® 647-conjugatedAffiniPure Goat anti-Armenian hamster IgG, Jackson Immunoresearch;1:1000, Alexa Fluor® 568 Goat anti-Rabbit IgG, A-11036, Thermo FisherScientific®; 1:500, DyLight™ 405 Goat anti-Rat IgG, JacksonImmunoresearch). After a 10 min PBS wash, organoid sections wereincubated for 5 min in PBS containing DAPI (1:10000, 62248, ThermoFisher Scientific®). The stained organoid sections were then washedthree times for 10 min in PBS, air dried and mounted with ProLong™ GoldAntifade Mountant (P36930, Thermo Fisher Scientific®).

Mouse Kidney IF Staining

Mice were anesthetized with 3 L/min O₂ mixed with 3% isoflurane usingthe Combi-vet® system (Rothacher Medical, Bern, Switzerland) for 5 min.Mouse kidneys were removed, sagittally cut to half and rapidly frozen inTissue-Tek® O.C.T. Compound, Sakura® Finetek (OCT) (25608-930, VWR)using dry ice and 100% ethanol. Five pm-thick sagittal cryosections(CM1950, Leica) were thaw mounted on microscope slides (Fisherbrand™Superfrost™ Plus, Fisher Scientific) and kept in the cryostat (at −26°C.) for the duration of the sectioning process. Sections wereimmersion-fixed for 10 min in 4% PFA (15710, Electron MicroscopySciences), then washed in PBS. The slides were then subjected to antigenretrieval by immersion in 10 mM citric acid buffer (pH=6), for 10minutes at 95° C. Following a wash in PBS, the slides were incubated for20 min at room temperature in PBS blocking solution containing 5% normalgoat serum (005-000-121, Jackson ImmunoResearch), 0.2% Triton X-100(X100-100ML, Sigma-Aldrich) and 2% bovine serum albumin (BSA)(A9576-50ML, Sigma-Aldrich). Sections were then incubated at 4° C.overnight with primary antibodies diluted in the same blocking solution(1:500, monoclonal Armenian hamster anti-MUC1, Ab80952, Abeam; 1:500,monoclonal Fab-A-V5H anti-MUC1-fs, AbD22655.4, Bio-Rad; 1:400,polyclonal Rabbit anti-Aquaporin 2, AQP-002, Alomone labs; 1:1000,polyclonal Rabbit anti-NCC, SPC-402, StressMarq; 1:300, monoclonalRabbit anti-ERp72, 5033, Cell signaling technology; 1:100, polyclonal,Rabbit anti-TMED9, 21620-1-AP, Proteintech). Stained sections wererinsed three times for 10 min in PBS and incubated with secondaryantibodies diluted in PBS containing 0.1% Triton X-100 for 2 hr at RT(1:500, Alexa Fluor® 488-conjugated AffiniPure F(ab′)2 Fragment Goatanti-Human IgG, 109-546-097, Jackson Immunoresearch; 1:500, Alexa Fluor®647-conjugated AffiniPure Goat anti-Armenian hamster IgG, 127-605-160,Jackson Immunoresearch; 1:1000, Alexa Fluor® 568 Goat anti-Rabbit IgG,A-11036, Thermo Fisher Scientific®). Following three washes of 10 min inPBS, stained sections were treated with 1:10000 DAPI solution (62248,Thermo Fisher Scientific®) in PBS for 5 min, washed three times for 10min in PBS, air dried and mounted using ProLong™ Gold Antifade Mountant(P36930, Thermo Fisher Scientific®).

Human Kidney Biopsies IF Staining

Paraffin sections were deparaffinated, hydrated and subjected to antigenretrieval by immersion in 10 mM citric acid buffer (pH=6). Endogenousperoxidase was blocked with 1% sodium azide and 0.3% H₂O₂ for 10 minutesfollowed by blocking with 5% fetal bovine serum (FBS) in PBS for 30minutes. Sections were then incubated at 4° C. overnight with primaryantibodies in 5% BSA in PBS (1:500, Alexa Fluor® 488-conjugated Fabfragment AbD2265454, anti-MUC1-fs, Bio-Rad; 1:100, monoclonal, Mouseanti-Epithelial Membrane Antigen (EMA), M061329-2, Dako; 1:100,polyclonal, Rabbit anti-TMED9, 21620-1-AP, Proteintech). Followingwashing, the sections were incubated for 1 hr at 37° C. with secondaryantibodies diluted in 5% BSA in PBS (1:500, Alexa Fluor® 555 Donkeyanti-Rabbit IgG, A-31572, Thermo Fisher Scientific®; 1:500, Alexa Fluor®647 Donkey anti-Mouse IgG, A-31571, Thermo Fisher Scientific®). Slideswere washed and mounted in ProLong Gold Antifade Mountant with DAPI(P36931, Thermo Fischer Scientific).

TUNEL Assay in Mouse Kidney Tissue

To detect apoptotic nuclei, tissue sections were prepared and fixed asdescribed above, permeabilized for 20 min with 0.2% Triton X-100(X100-100ML, Sigma-Aldrich) and stained using terminal deoxy transferaseuridine triphosphate nick-end labeling (TUNEL) technique (G3250,promega), following the manufacturer's protocol. Stained sections werewashed and treated with 1:10000 DAPI solution (62248, Thermo FisherScientific®) in PBS for 5 min. Following three washes of 10 min in PBS,the sections were air dried and mounted using ProLong™ Gold AntifadeMountant (P36930, Thermo Fisher Scientific®).

Fluorescence Image Acquisition

All fluorescence imaging performed herein was done using the OperaPhenix High-Content Screening System (HH14000000, PerkinElmer). Forfluorescence imaging of cells (live cell or fixed cell imaging),CellCarrier Ultra microplates (either 96 well 6055302 or 384 well6057308, Perkin Elmer) were used, and a minimum of nine fields wereacquired per well using 20× or 63× water immersion objectives in aconfocal mode. For kidney organoids and mouse kidney section imaging,microscope slides (Fisherbrand™ Superfrost™ Plus, Fisher Scientific)were used. The entire specimen was first imaged for DAPI at 5× using thePreciScan™ feature (Perkin Elmer) to identify tissue. Pre-identifiedtissue regions were then imaged at higher resolution (20× or 63× waterimmersion objectives, confocal mode).

Fluorescence Image Analysis

Image analysis for all imaging experiments was performed using theHarmony software (PerkinElmer).

Image Analysis of IF Cell Staining

Cell nuclei were first identified using Hoechst staining, and cellnumber was calculated. Cytoplasmic regions were then detected aroundeach nucleus based on combined channels. The cells from the edge of thefield were eliminated from the analysis. For the quantification ofprotein abundance, the total signal intensity value for each antibodywas calculated separately in the cell cytoplasm and the average signalper cell was calculated for each well. For quantitation of MUC1-fscellular distribution and trafficking, the analysis was performed asdescribed in FIG. 13A using the “spot” identification feature for thedetection of MUC1-fs and the different organelles.

Live Cell Image Analysis

Caspase 3/7 activation and/or DRAQ7 staining were used to calculate thefraction of cells going through apoptosis and/or cell death,respectively. Single cells were first identified using the digital phasecontrast channel and cell number was calculated. Fluorescenceintensities were then measured and the threshold for Caspase 3/7 andDRAQ7 positive staining was determined. As an output, the fraction oflive (neither caspase3/7 nor DRAQ7 signal detected), apoptotic(caspase3/7 positive) or dead cells (DRAQ7 positive) was calculated ineach well at a particular time point.

For the measurement of P cell rescue from THP-induced cell death byBRD-4780 (FIGS. 3I and 3J), no dyes were added to avoid any influence oncell growth. Digital phase contrast images were used for cellidentification and count, and autofluorescence in the far-red channelwas used for detection of dead cells. Cells with no autofluorescencewere identified and calculated as live cell.

For the GFP-rhodopsin experiments (FIGS. 15D and 15E), cells wereidentified by digital phase contrast. GFP signal was calculated only incells expressing GFP signal above a minimal background (to identifysuccessfully transfected cells) and the mean intensity was averaged foreach well.

For GFP-Huntingtin experiments (FIGS. 15G and 15H), GFP signal in eachcell was calculated using the “spot” identification feature, and totalspot intensity for single cells was calculated and averaged for eachwell.

Image Analysis for Mouse Kidney Sections

MUC1-fs and MUC1-wt mean intensities were calculated in kidney sectionsof mice treated with either vehicle or BRD-4780 (FIGS. 4A, 4B and 12A).As MUC1-fs and MUC1-wt levels varied in different kidney regions, and assections might contain different portions of the different kidneyregions, the levels of these proteins were analyzed only in NCC-positivedistal convoluted tubules. To this end, single cell nuclei were firstidentified using DAPI channel, followed by cytoplasm detection using allcombined channels, excluding the nucleus. Each fluorescent channelintensity was measured and a threshold was set for the identification ofNCC positive cells. MUC1-fs and MUC1-wt levels were then calculated onlyin the NCC positive cells.

For apoptosis detection using TUNEL staining (FIG. 2G), TUNEL signal wascalculated in each nucleus of the section (excluding the tissueperiphery) and the threshold for TUNEL-positive cells was established.Total number of apoptotic cells was calculated as the number of nucleiin which TUNEL signal exceeded the threshold level. This number wasnormalized (divided) to the total number of nuclei in the entire tissuesection (FIGS. 2G and 2H). For visualization and validation of tissueimage analysis, single cells depicted by the analysis were plotted as ascatter plot according to their position on the slide using Spotfiresoftware allowing thus to observe a tissue architecture and positioningapoptotic cells within their relative location. The identified TUNELpositive cells were highlighted by a red color.

Image Analysis for Mouse Retinal Sections

Rhodopsin intensity was calculated in retinal sections of Rho/+ micetreated with either vehicle or BRD-4780 (FIGS. 21A and 21B), as comparedto wild-type mice treated with vehicle alone. In healthy retina,Rhodopsin primarily localized to the outer segment of the photoreceptors(OS, indicated by the arrow in FIG. 21A), instead of localizing aroundthe photoreceptor nuclei in the outer nuclear layer (ONL, indicated byan asterisk in FIG. 21A). Since there is no Rhodopsin-P23H specificantibody, the amount of Rhodopsin staining in the outer nuclear layerwas calculated as a measurement of Rhodopsin accumulation inintracellular compartments. The calculated Rhodopsin staining in the ONLwas normalized to the nuclear staining, to correct for differences incell number.

Image Analysis for Organoids

For a sequence analysis illustration, refer to FIG. 12B. Tubularstructures expressing MUC1 proteins were identified and selected usingMUC1-wt positive staining. First, a reference region was generatedaccording to MUC1-wt positive staining in order to depict the apicalpart of each tubule. Subsequently, the intratubular region was definedbased on the reference region with the addition of 20 μm to expand thisregion in order to capture the entire tubule. Laminin signal was thenused to exclude the extracellular basal space. Finally, MUC1-fs signalwas depicted within this intratubular area using the “spot”identification feature, and mean intensity of this signal was averagedfor entire organoid section. MUC1-wt signal intensities were calculatedwithin each WT reference region and averaged for entire section.

High Content Screening

For high content screening an automated system was used, consisted ofrobotic arms; plate stackers; a HighRes Pin Tool; Liconic incubators;Biotek plate washers; dedicated Thermo Fisher Combi Multidrop dispensersfor each assay reagent; and PerkinElmer High Content Imaging InstrumentOpera Phenix, all choreographed by Cellario software. Cell fixation andimmunostaining were all performed in a custom-designed light-protectedhood (HighRes Biosolutions). Data analysis and representation wasperformed using Genedata Screener (Genedata AG.) and Spotfire (TIBCO).

MUC1-fs IF Screen

P cells were seeded 24 hr prior to compound treatment at a density of12,000 cells/well in 384 well Cell Carrier Ultra plates (6057308, PerkinElmer), pre-coated with 0.25 mg/mL Synthemax II SC Substrate (3535,Corning). Compounds of the repurposing library set (Corsello et al.,2017) were used at 5 doses (35, 3.5, 0.35, 0.035 and 0.0035 μM) for theprimary screen and 10 doses (16, 5.6, 1.8, 0.6, 0.21, 0.07, 0.02, 0.008,0.002 and 0.0008 μM) for the following screens. The compounds, in tworeplicates, were transferred from compound source plates to the cellplates using the HighRes Pin Tool. DMSO was used as a negative controland JQ1 (250 nM) (a bromodomain inhibitor) was chosen as a positivecontrol, based on earlier studies showing its potent effect on reducingtotal MUC1 mRNA levels (data not shown). After 48 hr incubation, cellswere fixed for 20 min in 4% PFA (Electron Microscopy Sciences) in PBS,washed twice, then permeabilized (10 min) with 0.5% Triton-X100(X100-100ML, Sigma-Aldrich) in PBS and washed once more. Cells wereblocked for 10 min at RT with Blocking solution (100 mM Tris HCL pH8;150 mM NaCL; 5 g/L Blocking Reagent [11096176001, Roche]), thenincubated 90 min at RT with one of the following primary antibodies inRoche Blocking solution: 1:500, monoclonal Fab-A-VSH anti-MUC1-fs,AbD22655.2, Bio-Rad; 1:2000, monoclonal mouse anti-MUC1 (214D4),05-652-KC, Millipore; 1:1000, monoclonal, Rabbit anti-GM130 (D6B1) XP,12480, Cell signaling technology. The primary antibody cocktail wasincubated at RT for 1.5 hr, followed by four PBS wash cycles. Thesecondary antibody cocktail contained four components that were allprepared at a 1:1000 dilution in the Roche blocking solution andconsisted of Alexa Fluor® 488-conjugated AffiniPure F(ab′)2 FragmentGoat anti-Human IgG, 109-546-097, Jackson Immunoresearch; Alexa Fluor®647-conjugated Goat anti-Rabbit IgG, A-21246, Thermo Fisher Scientific®;Alexa Fluor® 546 Goat anti-mouse IgG, A-21123, Thermo Fisher Scientific®and Hoechst 33342 stain (62249, Thermo Fisher Scientific®). Thesecondary antibody cocktail was incubated at RT for 45 min, followed byfour PBS wash cycles. Finally, plates were sealed with a Plate Loc plateand stored in Liconic incubator at 10° C. until imaging.

Image acquisition and analysis was done as described elsewhere herein.Following image analysis, three parameters were selected, i) MUC1-fs andii) MUC1-wt total cytoplasm intensity (averaged per cell) and iii) cellnumber as was detected by Hoechst 33342 stained nuclei. The levels ofMUC1-fs and MUC1-wt found following DMSO and JQ1 were defined as 0 and−100% activity, respectively. The values obtained for all othercompounds, including BRD-4780 were normalized accordingly. Cell numberwas normalized to DMSO control. All compound concentrationsshowing >−20% reduction in cell number were masked out. Based on ±3median absolute deviation value (FIG. 10A), hit calling criteria for theprimary and secondary screens were chosen as MUC1-fs reduction>30% intwo or more consecutive concentrations for both replicates. For thesecondary screen, dose response curves were generated for each parameterusing Genedata Screener (Genedata AG.), and positive hits for theprofiling screens were selected based on the compounds activity forreducing MUC1-fs and lack of toxicity. MUC1-fs specificity (according toMUC1 fs/wt ratio) was used as an additional positive criterion forselection.

RT-PCR Profiling Screen

qPCR was performed using a previously described protocol (Bittker,2012). P cells seeded at 2000 cells/well in 384-well, clear bottom,white wall plates were grown for 24 hr, then treated with profilingcompounds transferred by pinning to duplicate plates. JQ1 (250 nM) andDMSO were used for controls as above. After 24 hr, cells were washed andcDNAs were made using ABI Cells-to-Ct kit (Thermo Fisher Scientific®,Waltham, Mass.). MUC1 and HMBS delta Cp values were determined using aRoche LightCycler 480 Instrument and TaqMan probes for MUC1 FAM (4351368assay ID Hs00159357_m1) and HMBS VIC (4448486-assay ID Hs00609297_m1)(Thermo Fisher Scientific®, Waltham, Mass.), in 5 μL reactions. The foldchange effect of the compounds on total MUC1 mRNA was normalized to JQ1and DMSO controls, as described above.

Viability Profiling Screen

P cells were seeded 12 hr prior to profiling compound treatment at adensity of 12,000 cells/well in 384 well Cell Carrier Ultra plates(6057308, Perkin Elmer), pre-coated with 0.25 mg/mL Synthemax II SCSubstrate (3535, Corning). After 24 hr, CellEvent Caspase-3/7 GreenDetection Reagent (C10423, Thermo Fisher Scientific®) and DRAQ7(DR71000, Biostatus) were added at 1:5000 final dilution in the presenceor absence of thapsigargin (100 nM). Cells were imaged daily during 7days to monitor viability. Image acquisition and analysis was done asdescribed above and viability was assessed as fraction of live cells atday 5 of thapsigargin treatment, and at the day 6 for DMSO.

Kidney Histology

Formalin-fixed, paraffin-embedded kidney sections of 4 μm thickness werestained with periodic acid-Schiff (PAS) by HMS Pathology Core. Lightmicroscopy PAS images were analyzed in a blinded fashion and classifiedusing standard criteria. Immunoperoxidase staining for MUC1-wt andMUC1-fs was performed by standard protocols using anti-MUC1-wt (ab80952,Abcam) and monoclonal Fab-A-V5H anti-MUC1-fs (AbD22655.2, Bio-Rad).HRP-linked goat anti-Armenian hamster (PA1-32045, Thermo FisherScientific®) was used to detect MUC1-wt and Mouse anti V5-HRP (R961-25,Thermo Fisher Scientific®) was used to detect MUC1-fs.

Western Blot and SDS-PAGE Gel Electrophoresis

Cells were lysed in lysing buffer solution (9803, Cell SignalingTechnology) containing protease inhibitors (05892791001, Roche) andphosphatase inhibitors (04906837001, Roche). Mouse kidney tissues werelysed by tissue homogenizer (Tissue-Tearor™, BioSpec Products) in lysingbuffer solution containing inhibitors as above, followed by 20 minrocking at 4° C. and centrifugation at 16,000 g, 4° C. for 5 min. Tonormalize protein concentration, proteins in the supernatant of cells orof kidney lysates were quantified using the Pierce BCA Protein Assay Kit(23225, Thermo Fisher Scientific®). Normalized protein lysates were thenmixed with NuPAGE LDS sample buffer (NP0008, Thermo Scientific) andNuPAGE reducing agent (NP0004, Thermo Scientific) and heated to 75° C.for 10 min prior to SDS-PAGE gel electrophoresis using NuPAGETris-Acetate SDS Running Buffer (LA0041, Thermo Scientific) or NuPAGEMES SDS running buffer (NP0002, Thermo Fisher Scientific®) depending onprotein molecular weight of interest. Electrophoretically separatedproteins were transferred to a nitrocellulose membrane (1704158, BioRad)using Trans-Blot® Turbo Blotting System (1704155, BioRad) followingmanufacturer's protocol. Membranes were blocked in 5% Nonfat Dry Milk(9999S, Cell Signaling Technology) in PBS with 0.1% Tween® 20 (PBS-T),and probed with primary antibody overnight at 4° C. Following threewashes with PBS-T, the membranes were incubated with secondary antibodyfor 1 hr at room temperature, washed three more times in PBS-T, andincubated with Super Signal West Dura (34076, Thermo Fisher Scientific®)or Super Signal West Pico (34087, Thermo Fisher Scientific®) andimmunoreactive bands were imaged by G:BOX Chemi XT4 (G:BOX-CHEMI-XT4,Syngene).

Cellular Thermal Shift Assay (CETSA)

CETSAs were performed as previously described (Jafari et al., 2014;Reinhard et al., 2015). In brief, P cells were treated in the presenceor absence of BRD-4780 for 1 hr, harvested (trypsinized), washed in PBS,resuspended in PBS (containing protease inhibitors) and distributed in0.2 ml PCR tubes (100 μl; 600,000 cells). Cells were incubated at theirdesignated temperatures for 3 min, then at 25° C. for 3 min and lysed bythe addition of 1% NP-40. Immediately thereafter, samples were snapfrozen and thawed using thermal cycler set at 25° C. Samples were spunat 20,000×g for 20 min (4° C.) to remove precipitated protein and thesupernatant was analyzed by Western blot to examine the TMED9/Nischarinthermal stability.

RNA Extraction from Cells

RNA was extracted from cells seeded onto 12-well plates using the RNeasykit (74004, Qiagen), following the manufacturer's protocol. RNA waseluted with Nuclease-Free water and total yield and purity of RNA wereassessed by NanoDrop™ 2000 (Thermo Fisher Scientific®).

RNA Extraction from Mouse Kidneys

RNA was extracted from a sagittal section of a half-kidney. Kidneytissue was homogenized using tissue homogenizer (Tissue-Tearor™, BioSpecProducts) in 1 mL of TRIzol™ Reagent (15596026, Thermo FisherScientific®). Following 5 min incubation, 0.2 mL chloroform(C2432-500ML, Sigma-Aldrich) was added, and samples were vigorouslymixed for 30 sec, then centrifuged at 12,000×g at 4° C. for 15 min. Theupper aqueous phase containing RNA was then vigorously mixed with 0.5 mLof isopropanol for 30 sec. After 10 min incubation at RT, the sampleswere centrifuged at 12,000×g at 4° C. for 10 min. The pellet wasresuspended in 1 mL of 75% ethanol and centrifuged at 12,000×g 4° C. for5 min. The RNA pellet was then air dried for 15 min, dissolved in 50 μLof Nuclease-Free Water (AM9937, Thermo Fisher Scientific®), treated withDNase I, Amplification Grade (18068015, Thermo Fisher Scientific®)following the manufacturer's protocol and assessed for yield and purityby NanoDrop™ 2000 (Thermo Fisher Scientific®).

RT-PCR Analysis of XBP-1 mRNA Splicing

XBP1 splicing was analyzed by standard RT-PCR. Briefly, RNA was isolatedfrom patient cells using a RNeasy Mini Kit (74106, Qiagen). One μg ofRNA was converted to cDNA using the SuperScript First-Strand Synthesissystem for RT-PCR (11904-018, Invitrogen). The primer sequences used areas follows: Human XBP1 Forward Primer: 5′ TTA CGA GAG AAA ACT CAT GGC C3′ (SEQ ID NO: 12). Human XBP1 Reverse Primer: 5′ GGG TCC AAG TTG TCCAGA ATG C 3′ (SEQ ID NO: 13). PCR was carried out on 3 μL of theresulting cDNA solution using the OneTaq Hot Start Master Mix (M0484S,NEB). Five μL of PCR product was run on a 2% agarose gel for 90 min at150 V.

cDNA Library Construction and Illumina® Sequencing

cDNA library construction and Illumina® sequencing were performed at theBroad Institute sequencing platform as follows. Concentrations ofpurified RNA were measured with the Quant-iT™ RiboGreen® RNA Assay Kit(Thermo Scientific #R11490) and normalized to 5 ng/μL. An automatedvariant of the Illumina® TruSeq™ Stranded mRNA Sample Preparation Kitwas used for library preparation from a 200 ng aliquot of RNA. Thismethod preserves strand orientation of the RNA transcript and uses oligodT beads to select mRNA from the total RNA sample. Following cDNAsynthesis and enrichment, cDNA libraries were quantified with qPCR usingthe KAPA Library Quantification Kit for Illumina® Sequencing Platformsand then pooled equimolarly. For Illumina® sequencing, pooled librarieswere normalized to 2 nM and denatured using 0.1 N NaOH prior tosequencing. Flow cell cluster amplification and sequencing wereperformed according to the manufacturer's protocols using either theHiSeq 2000 or HiSeq 2500. Each run was a 101 bp paired-end with aneight-base index barcode read. Data was analyzed using the BroadInstitute Picard Pipeline, which includes de-multiplexing and dataaggregation.

UPR Branch Activation

The involvement of the UPR in MUC1-fs induced cytotoxicity was detectedin N and P cells using immunoblot or IF. For IF experiments (FIGS. 2B,9I and 9J), N and P cells were plated at 9,000 and 6, 000 cells/wellrespectively and cultured for 24 hr. Cells were pretreated with eitherDMSO or 10 μM inhibitors of ATF6 (PF-429242;SML0667, Sigma-Aldrich),PERK (GSK2656157;5046510001, Sigma-Aldrich) and IRE (4g8C;1902, AxonMedchem) for 1 hr, followed by application of different concentrationsof THP. Cell viability was determined using caspase activation asdescribed above. For immunoblot experiment (FIGS. 2C and 2D), N and Pcells were grown in 6-well plates to high confluence and treated withDMSO or THP (100 nM) for 12 hr. The cells were then processed forimmunoblot analysis as described above.

BRD-4780 Treatment in Mice

The effect of BRD-4780 on MUC1-fs levels in mouse kidney was tested inage-matched fs/+ and wt/+ male mice (27-38 weeks old). BRD-4780 (1, 10and 50 mg/Kg/day) or vehicle (PBS) were administered daily by oralgavage for 7 days. Animal weight was observed daily to monitor toxicity.On day 7, mice anesthetized for 5 min with 3 L/min 3% isoflurane in Ozusing the Combi-vet® system (Rothacher Medical, Bern, Switzerland) weresacrificed, and kidneys were removed for further analysis.

BRD-4780 Treatment in Rho-P23H Mice

The effect of BRD-4780 on Rhodopsin accumulation in intracellularcompartments of mouse retinae were tested in age-matched Rho-P23H/+ and+/+ male mice. BRD-4780 (50 mg/Kg/day) or vehicle (PBS) wereadministered daily by oral gavage for 28 days, starting at PND28. Animalweight was observed daily to monitor toxicity. On day 28, mice weresacrificed, and eyes were removed for further analysis.

BRD-4780 Treatment in Kidney Organoids

Day 25 kidney organoids were transferred from Transwell membranes tolow-attachment Corning® Costar®) TC-Treated 24-Well Plates (3527,Sigma-Aldrich) in 250 μL STEMdiff™ APEL™2 Medium (05270, Stem cellTechnologies), then treated with 10 μM BRD-4780 or DMSO vehicle for 72hr at 37° C. After 72 hr, organoids were washed twice with PBS andprocessed for IF detection.

BRD-4780 Treatment in Cells

Detection of MUC1-fs in P cells at baseline and following secretorypathway perturbations was performed using immunoblot and IF analyses.For immunoblot experiments (FIGS. 5E and 13D), P cells were grown in6-well plates to high confluence. For IF experiments (FIGS. 5B-5D and13A-13C), P cells were plated in 384-well plates at 12,000 cells/well.Following 24 hr, cells were treated with 5 μM BRD-4780 or DMSO alone, orin combination with 100 nM Bafilomycin A (B1793, Sigma-Aldrich), or 200ng/mL Brefeldin A (B7651, Sigma-Aldrich) for a maximum period of 24 hras indicated. For TMED9 or Nischarin depletion experiments,transduced/transfected P cells were plated either in 96-well plates at60,000 cells/well for IF (FIGS. 6C, 6D and 14B), or grown in 6-wellplates to high confluence for immunoblot detection (FIGS. 6B and 14C).Cells were then treated with 5 μM BRD-4780 or DMSO for 24 hr (for IFdetection) or 72 hr (for immunoblot analysis) and were processedaccordingly. AtT-20 cells stably transduced with C126R mutant UMODplasmid were treated for 72 hr with 10 μM of BRD-4780 or vehicle DMSO.For thapsigargin treatment, AtT-20 cells pretreated 24 hr with 1 or 10μM of BRD-4780 were exposed to 10 nM of thapsigargin for 48 hr. Thecells were either imaged for IF detection of UMOD (FIGS. 15A and 15B) orprocessed for immunoblot analysis. N cells transiently expressingP23H-GFP mutant rhodopsin were plated in 96-well plates at 20,000cells/well and grown for 24 hr, then DMSO or 5 μM BRD-4780 were added tothe medium as a pre-treatment. Following 48 hr, cells in the presence orabsence of BRD-4780, were transfected with P23H mutant rhodopsin-GFPplasmid. Eight hr post-transfection, the cells were washed once andmedia containing DMSO or 5 μM BRD-4780 was added. Plates were imaged 24hr following transfection.

HEK293T stably expressing huntingtin 97Q-GFP were treated with 10 μM ofBRD-4780 or vehicle DMSO, then monitored for GFP intensity by live cellimaging.

Cell Treatment for RNA-Seq Experiments

For RNA-seq experiments, N and P cells were plated in 12-well plates at200,000 cells/well. Subsequently, 1 μM of BRD-4780 or DMSO were appliedfor 24 hr followed by 12 hr exposure to 100 nM of thapsigargin (THP) orDMSO for control. At the end of the experiment, RNA was extracted fromtreated cells and sequencing was performed as described below.

Absorption, Distribution, Metabolism, and Excretion (ADME) of BRD-4780

PBS Solubility

Compound solubility was determined in PBS at pH 7.4. Each compound wasprepared in triplicate at 500 μM in both 100% DMSO and 100% PBS bydrying down 10 μL of a 10 mM DMSO compound stock solution and thenadding 200 μL of PBS. Compounds were allowed to equilibrate at roomtemperature with a 750 rpm vortex shake for 18 hr. Prior toequilibration, StirStix were added to each well to aid in the preventionof aggregation. After equilibration, samples were centrifuged (32×G) toremove undissolved particulates and a 20 μL aliquot of supernatant wasdiluted with 480 μL acetonitrile. The resulting solution was analyzed byUPLC-MS/MS with compounds detected by SIR detection on a singlequadrupole mass spectrometer. The peak areas of the 100% DMSO sampleswere used to create a two-point calibration curve to which the peak arearesponse in PBS was fit. Solubility in PBS was calculated using thefollowing equation: Conc. (PBS)=Conc. (DMSO)×[area (PBS)/area (DMSO)].(LC System, Waters Acquity H-Class; MS System, Waters Acquity SQDetector; Column, Waters Acquity UPLC BEH C18, 1.7 μm, 1.0×50 mm; MobilePhase A, Water with 0.1% Ammonium Hydroxide (or 0.5% trifluoroaceticacid); Mobile Phase B, Acetonitrile with 0.1% Ammonium Hydroxide (or0.6% trifluoroacetic acid); Flow Rate, 0.45 mL/min; Column Temperature,60° C.).

Plasma Stability

Plasma stability was determined at 37° C. at 5 hr in plasma. Eachcompound was prepared in duplicate at 5 μM in plasma diluted 50/50 (v/v)with PBS pH 7.4 (0.95% acetonitrile, 0.05% DMSO). Duplicate plates wereprepared. One plate was incubated at 37° C. for 5 hr with a 350 rpmorbital shake, while the other plate was immediately quenched. Each wellwas quenched by adding acetonitrile to a 3:1 ratio (v/v, ACN/plasma).After quenching, samples were centrifuged (32×G) to pellet precipitatedparticulates and an aliquot of supernatant was diluted 50/50 (v/v) withwater. The resulting solution was analyzed by UPLC-MS/MS with compoundsdetected by MRM detection on a triple quadrupole mass spectrometer. Thecompound peak areas at 0 and 5 hr were compared to determine a percentremaining. Percent remaining in plasma was calculated through thefollowing equation: % remaining=[area (5 hr)/area (0 hr)]×100. (LCSystem, Waters Acquity I-Class; MS System, AB Sciex Triple Quad 4500;Column, Waters Acquity UPLC BEH C18, 1.7 μm, 2.1×50 mm; Mobile Phase A,Water with 0.1% formic acid; Mobile Phase B, Acetonitrile with 0.1%formic acid; Flow Rate, 0.90 mL/min; Column Temperature, 55° C.).

Plasma Protein Binding

Plasma protein binding was determined by equilibrium dialysis using aRapid Equilibrium Dialysis (RED) device (Pierce Biotechnology). Eachcompound was prepared in duplicate at 5 μM in plasma (0.95%acetonitrile, 0.05% DMSO) and added to one side of the membrane with PBSpH 7.4 added to the other side. Compounds were incubated at 37° C. for 5hr with a 350 rpm orbital shake. After incubation, an aliquot was takenfrom each side of the membrane and quenched by adding acetontrile to a3:1 ratio (v/v, ACN:plasma). After quenching, samples were centrifuged(32×G) to pellet precipitated particulates and an aliquot of supernatantwas diluted 50/50 (v/v) with water. The resulting solution was analyzedby UPLC-MS/MS with compounds detected by MRM detection on a triplequadrupole mass spectrometer. The compound peak area on the buffer sideof the membrane was compared to the peak area on the plasma side of themembrane to determine percent bound. Percent bound in plasma wascalculated through the following equations: % free=[area (buffer)/area(plasma)]×100. % bound=100−% free. (LC System, Waters Acquity I-Class;MS System, AB Sciex Triple Quad 4500; Column, Waters Acquity UPLC BEH08, 1.7 μm, 2.1×50 mm; Mobile Phase A, Water with 0.1% formic acid;Mobile Phase B, Acetonitrile with 0.1% formic acid; Flow Rate, 0.90mL/min; Column Temperature, 55° C.).

Liver Microsomes Stability

Stability was determined at 37° C. at 1 hour in liver microsomes. Eachcompound was prepared in duplicate at 1 μM in liver microsomes diluted50/50 (v/v) with PBS pH 7.4. After addition of compounds to microsomemixture, the plate was sealed and vortexed and 100 μL of each sample wasadded to 300 μL acetonitrile (with internal standard). Assay plate wasincubated at 37° C. for 1 hr with a 350 rpm orbital shake, and followingthe hour 100 μL of each sample was added to 300 μL acetonitrile (withinternal standard). After quenching, samples were refrigerated for atleast 1 hour. The samples were centrifuged (32×G) to pellet precipitatedparticulates and an aliquot of supernatant was diluted 50/50 (v/v) withwater. The resulting solution was analyzed by UPLC-MS/MS with compoundsdetected by MRM detection on a triple quadrupole mass spectrometer. Thecompound peak areas in the 0 and 1 hour samples, -NADPH sample, and nocompound control sample were compared to determine a percent remaining.Percent remaining in microsomes was calculated through the followingequation: % remaining=[area (1 hr)/area (0 hr)]×100. (LC System, WatersAcquity I-Class; MS System, AB Sciex Triple Quad 4500; Column, WatersAcquity UPLC BEH C18, 1.7 μm, 2.1×50 mm; Mobile Phase A, Water with 0.1%formic acid; Mobile Phase B, Acetonitrile with 0.1% formic acid; FlowRate, 0.90 mL/min; Column Temperature, 55° C.).

RNA-Seq Analysis

Data processing and statistical analyses for all RNA-seq experimentswere performed in RStudio—Version 1.0.153 as follows. First, barn filesobtained from the Broad Institute sequencing platform were reverted toFASTQ files followed by alignment of paired end reads to the mm10 mouseor hg19 human reference genome using STAR. Quality control metrics wereobtained using RNA-SeQC and expression levels were estimated by runningRSEM with default parameters on these alignments. RSEM's gene levelexpression estimates were normalized according to edgeR for sequencingdepth and multiplied by 1,000,000, to obtain counts per million (CPM).Genes with CPM<1 in fewer than three samples (number of replicates) wereremoved from further analysis. TMM normalization was applied to thedata, accounting for gene composition. For further downstream analysis(e.g. boxplots) the data was log 2-transformed. In mouse kidney RNA-seqexperiments, two samples were removed from the analysis (fs/+ treatedwith BRD-4780 replicate 3 and +/+ treated with BRD-4780 replicate 1) dueto significantly low library sizes. For statistical testing, generalizedlinear models obtained in edgeR were applied with the assumption ofnegative binomial distribution with default parameters using theexpected counts from RSEM followed by likelihood ratio test. Genes wereconsidered to be significantly differentially expressed using asignificance level of α=0.05 following Benjamini Hochberg correction formultiple hypothesis testing.

The Functional Annotation Tool of DAVID was used to detect significantsignatures differentially downregulated in fs/+ mice following BRD-4780treatment. Differentially downregulated genes were analyzed using GOterms of Biological Processes. The reported p-value is a modified FisherExact p-value (EASE score) and a gene set was considered enriched if asignificance level of α=0.05 was reached following Benjamini Hochbergcorrection.

UPR Specific Transcriptome

UPR branch activation analysis is presented as boxplots. Each box of theboxplot consists of genes comprising an individual UPR branch (Adamsonet al., 2016). The expression of each gene in the group is plotted asmean of the scaled expression profiles obtained from three replicates.

Statistical Analysis

Statistical analysis was performed and presented using Graphpad Prismversion 7.0 software. All data is presented as means±standard deviationunless otherwise specified. Statistical comparisons of two groups for asingle variable with normal distributions were analyzed by unpairedt-test. Statistical comparisons of two or more groups with oneindependent variables were analyzed by One-way ANOVA with Tukeypost-tests. Statistical comparisons of two or more groups with twoindependent variables were analyzed by Two-way ANOVA with Tukeypost-tests. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001.

Example 2: Mutant MUC1-fs is Retained Intracellularly in TubularEpithelial Cells

The location of the wild-type (MUC1-wt) and mutant (MUC1-fs) protein ina kidney biopsy from a 50-year-old MKD patient, who was a heterozygouscarrier for a C insertion frameshift mutation (MUC1^(wt)/MUC1^(fs)) wascharacterized. As compared to normal kidney (FIG. 1A, left), thepatient's tissue showed tubular atrophy, tubular dilation and someinterstitial fibrosis (FIG. 1A, right). The MUC1-wt and MUC1-fs proteinwere visualized by immunoperoxidase staining with wild-type- andmutant-specific antibodies in the same kidney biopsy. MUC1-wt maintainedits normal localization at the apical membrane of distal tubules andcollecting ducts, whereas MUC1-fs showed intracellular accumulation(FIG. 1B).

Heterozygous knock-in mice in which one of the normal mouse mMuc1^(wt)(+) alleles was replaced with either a human wild-type (hMUC1^(wt) orwt) or mutant (hMUC1^(fs) or fs) allele (FIGS. 7A and 7B) weregenerated. The knock-in mice have been indicated as wt/+ or fs/+ ascompared to +/+ for the parental wild-type mouse line for clarity. Theexpression of the corresponding human protein was confirmed usingWestern blot (FIG. 7C) and immunohistochemistry (IHC) (FIG. 7D). IHC infs/+ heterozygous mice demonstrated that the distribution of the normaland mutant human MUC1 proteins was consistent with the pattern seen inkidney biopsies from MKD patients (FIG. 1B): normal MUC1-wt protein wasfound at the plasma membrane of tubular epithelial cells and the mutantMUC1-fs protein was found intracellularly (FIG. 1D).

While no pathologic changes were detected in kidneys from the parentalstrain (+/+) or mice carrying the normal human allele (wt/+) (FIG. 1C),mice carrying the mutant allele (fs/+) developed progressive kidneydisease (FIG. 1E). The pathological changes detected in fs/+ mice weresimilar to those observed in MKD patient biopsies, marked by tubulardilations and some interstitial fibrosis (FIG. 1E). This kidneypathology developed as early as 8 months of age in female fs/+ mice(FIG. 1E) compared to male mice in which tubular pathology was noted at12 months of age (FIG. 7F). Serum creatinine was mildly elevated infemale fs/+ mice at 76 weeks of life and beyond, but not in males (FIG.7E and FIG. 17). The distribution of normal mouse and mutant human MUC1proteins in different segments of the kidney was characterized by doublelabeling immunofluorescence (IF) microscopy with segment-specificmarkers (FIG. 8A). Both MUC1-fs and MUC1-wt were expressed insodium-chloride symporter (NCC or SLC12A3)-positive distal convolutedtubules and aquaporin 2 (AQP2)-positive collecting ducts in the cortexand inner medulla, whereas Lotus tetragonolobus lectin (LTL)-positiveproximal tubules in the outer medulla expressed solely MUC1-fs (FIG.8A). This finding was of interest because the outer medulla is thekidney region in which the mutant mice exhibited the earliest andultimately most severe histopathological changes (FIGS. 1E and 8B).

Human kidney organoids were generated (Morizane and Bonventre, 2016;Takasato et al., 2016) from MKD patient iPSCs. The iPSCs were made fromerythroblasts obtained from three MKD patients. iPSCs from unaffectedsiblings served as controls. Mature organoids (29 day, see Example 1above) developed nephron structures including proximal and distaltubules, which were recently characterized in detail (Subramanian etal., 2019). MUC1-wt was detected in normal, sibling-derived andpatient-derived kidney organoids, at highest abundance in distal nephronstructures (FIG. 9A). In line with previous observations in MKD patientkidney biopsies (Bleyer et al., 2017; Yu et al., 2018), MUC1-fs wasobserved exclusively in MKD patient-derived organoids, in E-cadherin andNa/K-ATPase-positive tubules, and to a lesser extent, in LTL-positivetubules (FIGS. 1F and 13A). Notably, MUC1-wt was located at the plasmamembrane of these tubules (FIG. 1F), whereas MUC1-fs was localizedintracellularly (with basolateral membranes defined by Na/K ATPasestaining; FIG. 1F). Thus, human organoids recapitulated the subcellularlocalization of MUC1-wt and MUC1-fs, as seen in human kidney biopsies.

To enable downstream mechanistic studies, an immortalized tubularepithelial cell line was generated from a patient with MKD (P cells) andcompared MUC1 expression to a cell line derived from a normal humankidney (N cells; FIGS. 9B and 9C). P cells expressed MUC1-wt on theplasma membrane (FIG. 1G), similar to N cells (FIG. 9B), whereas MUC1-fswas found exclusively in P cells (FIGS. 9B and 9C) in diffuseintracellular, perinuclear pattern similar to that previously seen inpatient kidney biopsies, iPSC-derived kidney organoids and knock-inmouse kidneys (FIG. 1G).

Example 3: MUC1-fs Accumulation Triggers the ATF6 Branch of the UPR

To decipher the molecular mechanism by which MUC1-fs accumulation altersepithelial cell function, the involvement of the unfolded proteinresponse (UPR), a prominent mechanism for the regulation of cellularproteostasis (Plate and Wiseman, 2017), was analyzed. First, to addressthe involvement of IRE1, PERK and ATF6 branches of the UPR (Walter andRon, 2011) in P cells, the RNA-Seq data from P and N cells usingpublished transcriptional signatures of either general orbranch-specific UPR activation (Adamson et al., 2016) was analyzed. Thisanalysis indicated general activation of the UPR (Complex), and clear,significant, upregulation of the ATF6 branch (FIG. 2A). Minimaltranscriptional changes were noted in the IRE1 and PERK branches (FIG.2A).

Since the activation of specific UPR branches can promote eithercytoprotective or pro-apoptotic outputs (Hetz, 2012), the involvement ofUPR branches in P cell viability was tested by inhibiting one branch ata time (FIGS. 9D-9F). MUC1-fs accumulation alone did not induceapoptosis (as measured by caspase 3/7 activity; FIGS. 2B and 9G).However, the inhibition of ATF6 resulted in increased apoptosis in Pcells compared to N cells (FIG. 2B), suggesting that the ATF6 UPR branchmight be specifically upregulated to protect P cells fromMUC1-fs-induced toxicity. Consistent with this, ATF6 inhibition alsocaused enhanced accumulation of MUC1-fs in P cells (FIG. 9H). IRE1inhibition induced apoptosis broadly, in line with its generalcytoprotective role (Ishikawa et al., 2019; Lin et al., 2007), with nosignificant difference between P and N cells (FIG. 2B). PERK inhibitionhad no effect on apoptosis (FIG. 2B). Together, and without wishing tobe bound by theory, these results indicate that ATF6 is activated tocounteract MUC1-fs accumulation and associated toxicity. These resultswere validated by measuring the abundance of the main downstream sensorproteins of the three UPR branches. Consistent with the RNA-Seq data(FIG. 2A), upregulation of BiP, a chaperone activated by all three UPRbranches (FIG. 2C), was observed. The activation of the ATF6 branch wasverified by the increased abundance of ERp72 and GRP94 (FIG. 2C). Inkeeping with the above results, minimal changes in ATF4, the maintranscription factor of the PERK pathway, or CHOP, the proapoptotictarget gene of ATF4 (FIG. 2C), were detected. XBP1 mRNA splicing, a keystep in the activation of the IRE1 pathway (FIG. 2C), was also notdetected.

Given the increased vulnerability of P cells to ATF6 inhibition, it wasreasoned that increased stress signaling through the activation of thePERK and IRE1 arms of the UPR may further promote tubular cell injury.Therefore, the effect of thapsigargin (THP), a well-known ER stressor,was tested on P cells, and such cells were discovered to exhibit highersusceptibility to apoptosis compared to N cells (FIGS. 9I and 9J).Consistent with this, treatment with THP triggered the pro-apoptoticPERK pathway specifically in P cells, as shown by increased ATF4 andCHOP abundance (FIG. 2D). XBP1 splicing was also noted in P cells aftertreatment with THP (FIG. 2D). Taken together, these results indicatedthat THP-induced activation of the UPR drives P cell apoptosis, therebydefining a cell-based assay valuable for downstream drug screeningefforts.

The UPR pathways were also relevant in vivo. In kidneys of fs/+ mice,the ATF6 marker ERp72 was most abundant in the same tubular structuresas MUC1-fs in the outer medulla (FIG. 2E). Furthermore, increasedprotein abundance of calreticulin, ERp72, GRP94 and CHOP in tissuelysates from kidneys of 12-month old fs/+ mice (FIG. 2F) were found. Theincreased abundance of pro-apoptotic CHOP was associated with increasedapoptosis in 12-month old mice, as evidenced by positive TUNEL stainingprimarily in the outer medulla of kidneys from fs/+ mice (FIGS. 2G and2H).

Example 4: BRD-4780 Selectively Clears Mutant but not Wild-Type MUC1

To identify compounds that can remove MUC1-fs, a high content screen(HCS) using an IF cell-based assay that could simultaneously assess theabundance of MUC1-wt, MUC1-fs and cell number in a 384-well formatutilizing a fully automated staining protocol and confocal imagingmicroscopy system (z′ score 0.35, FIG. 10A) was developed. The BroadRepurposing Library, a set of 3713 compounds at different stages ofpreclinical and clinical development (Corsello et al., 2017) (FIGS. 3Aand 10A) was screened. The bromodomain inhibitor JQ1 was used as apositive control because preliminary in vitro experiments in P cellsshowed that it results in 100% transcriptional suppression of bothMUC1-fs and MUC1-wt.

For the primary screen, the Repurposing Library was tested at 5 doses(FIGS. 3A and 3B), with positive hits defined by (i) reduction ofMUC1-fs by >30% at a minimum of two consecutive compound doses, and (ii)lack of cell toxicity at these doses (no significant reduction in cellnumber). A total of 203 compounds (5%) met these criteria (FIG. 3B).These compounds were retested in a secondary screen, generatingdose-response curves at 10 doses and defining positive hits based on thesame two criteria as for the primary screen. A total of 71 compoundswere selected for further evaluation (FIGS. 3A and 3C). While many ofthe compounds caused comparable reduction of both MUC1-fs and MUC1-wt,it was noticed that some preferentially removed MUC1-fs (FIGS. 3B and3C). Furthermore, the 71 compounds were characterized utilizing threeassays: (i) the 10-point dose-response curve to test for compoundspecificity for MUC1-fs reduction (FIG. 3D) was re-measured; (ii) MUC1mRNA levels to eliminate compounds that reduced MUC1 protein abundancethrough transcriptional suppression were measured (MUC1-wt and MUC1-fsmRNAs are not readily distinguished and are transcribed from the samepromoter; FIG. 3E), and (iii) the ability of the compounds to rescue Pcells from cell death caused by THP (FIG. 3F) was tested.

A single compound, BRD-4780, emerged from these three profiling assays(FIG. 3A). First, BRD-4780 resulted in dose-dependent removal (EC50=143nM) of mutant MUC1-fs without decreasing wild-type MUC1 (FIGS. 3G and3H). Second, BRD-4780 showed no effect on MUC1 transcriptionalregulation while retaining efficacy in specifically removing mutantMUC1-fs protein in a dose responsive manner (FIG. 3E). Third, BRD-4780rescued P cells from THP-induced cell death (FIGS. 3I and 3J) andsignificantly reduced UPR activation (FIG. 10B). Taken together, thehigh-content screening and profiling assays identified BRD-4780 as acompound that selectively reduced mutant but not wild-type MUC1.

Example 5: BRD-4780 Removes Mutant MUC1-fs from Kidneys of HeterozygousKnock-in Mice

The ability of BRD-4780 to reverse the accumulation of MUC1-fs in vivowas then tested. Based on PK studies in 129S2 mice (FIGS. 11A-11F), thecompound (1, 10 and 50 mg/kg) or vehicle was administered to 8 month-oldmale heterozygous knock-in (fs/+) mice by daily oral gavage for 7 days.Following treatment, mice were sacrificed and their kidneys wereharvested and processed to assess MUC1-fs protein abundance by IF (FIGS.4A and 4B) and Western blot (FIG. 4C). BRD-4780 treatment resulted in adose-dependent removal of mutant MUC1-fs protein from mouse kidneys(FIGS. 4B and 4C). At the highest dose (50 mg/kg), efficient removal ofmutant MUC1-fs was noticed such that the tissue appeared nearlyindistinguishable from control (+/+) mouse kidneys (FIG. 4A). Consistentwith in vitro data, BRD-4780 had no effect on MUC1-wt (FIGS. 4A and12A). In addition to removing the toxic mutant protein, BRD-4780treatment downregulated pathways associated with ER stress and the UPR,as shown by pathway analysis of bulk RNA-Seq data (FIG. 4D; see Example1 above).

Example 6: BRD-4780 Removes MUC1-fs Protein from Patient iPSC-DerivedKidney Organoids

To confirm the human relevance of the above findings, the effect ofBRD-4780 on MUC1-fs was evaluated in kidney organoids generated fromiPSCs of patients with MKD. Using single cell genomics and IF, acomprehensive characterization of iPSC-derived human kidney organoidswas reported, and their reproducibility and potential utility for drugdiscovery was confirmed (Subramanian et al., 2019). Herein, the effectof BRD-4780 on MUC1-fs protein levels in kidney organoids derived fromiPSCs of three patients with MKD (P1-P3) compared to organoids derivedfrom an unaffected control (N1) was tested. BRD-4780 cleared the mutantprotein from intracellular compartments in all patient organoids (FIGS.4E, 4F and 12B), while MUC1-wt protein levels in patient or controlorganoids remained unchanged (FIGS. 4E and 4G). These resultssubstantiated the human relevance of the above-described findings.

Example 7: MUC1-fs Accumulates in the Early Secretory Pathway, in aTMED9 Cargo Receptor-Positive Compartment

MUC1-wt is a transmembrane glycoprotein, with a signal peptide (SP; FIG.7B) that directs it to the secretory pathway (Nath and Mukherjee, 2014).Newly synthesized MUC1-wt is transported from the ER to the Golgiapparatus for O-glycosylation prior to its delivery to the apical plasmamembrane (Apostolopoulos et al., 2015). Like all transmembrane proteins,MUC1-wt is packaged into COPII vesicles and is transported from the ERto the Golgi apparatus (Gomez-Navarro and Miller, 2016). At this point,cells can distinguish between wild-type and mutant proteins, ensuringthat only appropriately folded and assembled cargo proteins undergoforward transport through the Golgi apparatus to the endosomalcompartment (Gomez-Navarro and Miller, 2016) (FIG. 5A). Retrogradetransport from the cis-Golgi to the ER ensures that immature proteincargoes or escaped ER resident proteins are efficiently transported backto the ER by COPI vesicles (Gomez-Navarro and Miller, 2016) (FIG. 5A).However, misfolded proteins can get trapped along the early secretorypathway, between the ER and Golgi compartments (Gomez-Navarro andMiller, 2016). Consistent with this picture, it was identified hereinthat MUC1-wt localized clearly and specifically to the plasma membranein P cells (FIG. 1G). In contrast, mutant MUC1-fs was found in apunctate pattern throughout the cytoplasm (FIG. 1G). It was thereforelikely that MUC1-fs was being trapped somewhere along the earlysecretory pathway.

To determine exactly where MUC1-fs was being retained, a comprehensiveco-localization study with markers of different compartments of thesecretory pathway was performed (illustration, FIGS. 5A, 5B, 13A and13B). MUC1-fs was substantially more abundant in GM130-positivecis-Golgi compartment and TMED9 cargo receptor-positive vesicles than inother compartments (FIGS. 5A and 5B). TMED9, a member of the p24 cargoreceptor family, facilitates packaging and transport between the ER andcis-Golgi compartments (Strating and Martens, 2009), and is thought toplay a critical role in COPI retrograde transport from the cis-Golgiback to the ER (Adolf et al., 2019; Beck et al., 2009). It was alsoverified that MUC1-fs co-localized in a vesicular pattern with TMED9 inP cells, as well as in tubular epithelial cells from fs/+ mouse kidneys,from a kidney biopsy of a patient with MKD and from patient-derivedkidney organoids (FIG. 5C). Taken together, these data across fourdifferent sources (human cells, kidney organoids, and kidney biopsy aswell as mouse kidney) show that MUC1-fs is preferentially co-distributedwith the cargo receptor TMED9.

Example 8: BRD-4780 Releases MUC1-fs from the Early SecretoryCompartment

To assess the effect of BRD-4780 treatment on MUC1-fs subcellulardistribution over time, the co-localization study was repeated in thepresence of BRD-4780 over a 5 hour time course (FIG. 5D). Compared tobaseline (FIG. 5B), MUC1-fs was reduced in the early secretorycompartment after treatment with BRD-4780, and was instead progressivelyassociated with the TGN46-positive trans-Golgi, the EEA1-positiveendosomal and the LAMP1-positive lysosomal compartments (FIG. 5D). Theseresults indicated that BRD-4780 promotes anterograde trafficking andlysosomal degradation of MUC1-fs by releasing it from the earlysecretory compartment, where it had been trapped in the absence ofBRD-4780.

Example 9: An Intact, Functional Secretory Pathway is Required forBRD-4780-Mediated Removal of MUC1-fs

To test whether targeting for lysosomal degradation is the mechanism bywhich BRD-4780 removes mutant MUC1-fs, anterograde trafficking andlysosomal degradation were disrupted before and after treatment withcompound. The vesicular transport between the ER and Golgi was firstblocked with Brefeldin A (BFA) (Chardin and McCormick, 1999). Thisresulted in the accumulation of an intermediate glycosylated 117 kDMUC1-fs protein in the ER (as compared to 100 kD MUC1-fs protein atbaseline; FIGS. 5E and 13C) (Bosshart et al., 1991). BFA also abrogatedthe effect of BRD-4780 on MUC1-fs clearance (FIG. 5E). Second,inhibition of lysosomal degradation by Bafilomycin A (Yoshimori et al.,1991) resulted in a 170 kD MUC1-fs protein retained in late secretorycompartments (trans-Golgi, late endosomes and lysosomes)(FIGS. 5E and13C). This 170 kD MUC1-fs protein was present but less abundant incontrol P cells at baseline, likely representing a fully O-glycosylatedversion of the protein (Apostolopoulos et al., 2015). Importantly,lysosomal inhibition with Bafilomycin A prevented BRD-4780 from clearingmutant MUC1-fs (FIG. 5E). Given that treatment with BFA or Bafilomycin Aalone resulted in accumulation of MUC1-fs, it was concluded thattrafficking through the secretory pathway to the lysosome is thefundamental mechanism for MUC1-fs degradation in P cells. In support ofthis, inhibition of the proteasome had no effect on MUC1-fs accumulationat baseline, or its removal by BRD-4780, reinforcing the conclusion thatMUC1-fs is degraded in the lysosome (and not the proteasome)(FIG. 13D).Taken together, these experiments established that the effect ofBRD-4780 on clearing MUC1-fs required a functional secretory pathway andlysosomal degradation.

Example 10: TMED9 is Upregulated in Kidney Cells Expressing MUC1-fs

Since MUC1-fs was found at highest abundance in TMED9- andGM130-positive compartments at baseline (FIG. 5B), the abundance ofTMED9 and GM130 was explored in tubular epithelial cells. GM130abundance was comparable between patient iPSC-derived kidney organoids(P1) relative to controls (N1), and was not affected after treatmentwith BRD-4780 (FIG. 14A). In contrast, TMED9 abundance was higher inpatient-derived organoids (P1), specifically in cells expressingMUC1-fs, compared to controls (N1). BRD-4780 treatment not only clearedMUC1-fs, but also reduced TMED9 to levels comparable to cells in N1organoids (FIG. 6A). This further supported the functional role of TMED9in the entrapment of mutant MUC1-fs in the early secretory pathway.

Example 11: TMED9 Deletion Phenocopies the Effect of BRD-4780 on MUC1-fsRemoval

To confirm the role of TMED9 in the mechanism of action of BRD-4780,CRISPR-Cas9 was used to delete TMED9 from P cells (FIG. 6B). Geneticdeletion of TMED9 phenocopied the effect of BRD-4780 and resulted in theremoval of MUC1-fs from P cells, as shown by Western blot (FIG. 6B) andIF (FIGS. 6C and 6D). Of note, the abundance of β-COP, an integralcomponent of COPI vesicles (Beck et al., 2009), was not affected eitherby TMED9 deletion or BRD-4780 treatment (FIG. 6B). Similarly, MUC1-wtabundance and its localization on the plasma membrane were not affectedby TMED9 deletion or BRD-4780 treatment (FIG. 14B). Thus, BRD-4780appears to work in a targeted fashion to remove the mutant protein cargoassociated with TMED9 without disrupting the cell's baseline transportmachinery.

Example 12: BRD-4780 Directly Binds its Molecular Target, TMED9

BRD-4780 was originally annotated as a selective ligand for theimidazoline-1 receptor (I1R) and studied as a potential centralanti-hypertensive therapy. However, due to lack of efficacy as ananti-hypertensive in several animal studies, BRD-4780 was never advancedinto the clinic (Munk et al., 1996). The protein nischarin has beenpreviously suggested as a candidate I1R (Nikolic and Agbaba, 2012;Piletz et al., 2000; Zhang and Abdel-Rahman, 2006). Therefore, it wasthen assessed herein if nischarin is involved in the mechanism of actionof BRD-4780 in clearing MUC1-fs. Neither RNAi-mediated depletion norCRISPR-Cas9-mediated deletion of nischarin in P cells had any effect onMUC1-fs protein abundance or on the efficacy of BRD-4780 (FIGS. 6C, 6Dand 14C), ruling out nischarin as a target of the compound. 17additional small molecules annotated as I1R ligands were tested and itwas found that none were active in removing MUC1-fs (FIG. 14D).Collectively, these findings indicated that BRD-4780 works through amolecular mechanism that does not involve nischarin/I1R.

The TMED9 cargo receptor (i) co-localized in the same vesicularcompartments as MUC1-fs at baseline, (ii) was upregulated in MUC1-fsexpressing kidney cells in patient-derived organoids, (iii) was reducedto baseline levels after BRD-4780 treatment and (iv) its deletionphenocopied the effect of BRD-4780. Suspecting TMED9 as a likelymolecular target of BRD-4780, evidence of direct drug-target engagementwas sought. A cellular thermal shift assay (CETSA, see Example 1 above)was performed (Jafari et al., 2014; Reinhard et al., 2015), in whichunbound proteins denature and precipitate at elevated temperatures,whereas drug-bound proteins remain in solution (Jafari et al., 2014).The CETSA for TMED9 in the presence of BRD-4780 demonstrated twofindings consistent with direct binding (FIG. 6E). First, BRD-4780shifted the TMED9 heat denaturation curve to significantly highertemperatures, and second, BRD-4780 also up-shifted the SDS-PAGEmigration of TMED9 at all temperatures, consistent with likely covalentmodification of TMED9 or other posttranslational modifications (FIG.6E). In contrast, the same experiment for nischarin/I1R revealed noevidence indicative of direct engagement with BRD-4780 (FIG. 14E). Inaggregate, these findings identified the cargo receptor TMED9 as amolecular target of BRD-4780. Furthermore, these data indicated aheretofore unknown mechanism of action for this compound—namely, thatBRD-4780 binding to TMED9 releases MUC1-fs from the early secretorycompartment, thereby promoting its anterograde trafficking intoendosomes and finally into lysosomes, where it can be degraded (FIG.6F).

Example 13: BRD-4780 is Effective in Removing Several Misfolded Proteins

Whether the effect of BRD-4780 was specific to the MUC1-fs protein (ascompared to MUC1-wt), or whether it might also facilitate the removal ofmisfolded proteins in other proteinopathies involvingmembrane-associated proteins was also examined. The compound was firsttested in a cellular model of another autosomal dominant proteinopathyof the kidney, uromodulin (UMOD)-associated kidney disease (Johnson etal., 2017; Schaeffer et al., 2017), a disorder with no availabletreatment. BRD-4780 was applied to AtT20 cells expressing a mutant C126RUMOD protein that accumulates intracellularly. Remarkably, BRD-4780reduced the levels of mutant UMOD protein as measured by IF (FIGS. 15Aand 15B) and confirmed by Western blot (FIG. 15C).

The ability of BRD-4780 to alleviate a proteinopathy outside the kidneywas also tested. Retinitis pigmentosa (RP), the most common inheritedretinal degenerative disease, is caused by mutations in rhodopsin(Athanasiou et al., 2018; Dryja and Li, 2017). Most rhodopsin mutations,including P23H, result in a misfolded protein that accumulatesintracellularly leading to photoreceptor cell death (Athanasiou et al.,2018). BRD-4780 was applied to N cells over-expressing GFP-taggedrhodopsin P23H and the effect of the compound was studied by followingGFP fluorescence over 24 hours. BRD-4780 produced significantlydecreased GFP fluorescence at 24 hours in GFP-rhodopsin P23H-expressingcells (FIGS. 15D and 15E). The viability of cells expressing P23H mutantsignificantly improved upon treatment with BRD-4780 (FIG. 15F).

Additional indication of the ability of BRD-4780 to treat RP wasobtained via observation of rhodopsin antibody staining of mouse retinalsections in Rho/+ mice treated with either vehicle or BRD-4780. Resultsof such staining experiments indicated that treatment with BRD-4780significantly reduced rhodopsin accumulation in intracellularcompartments, likely reflective of an alleviation of the rhodopsinproteinopathy present in these mice (FIGS. 21A and 21B).

To see if the effect of BRD-4780 was restricted to misfolded proteinswhose wild-type versions are membrane-associated (as is the case forMUC1, UMOD and rhodopsin)(Athanasiou et al., 2018; Hilkens and Buijs,1988; Johnson et al., 2017; Litvinov and Hilkens, 1993; Schaeffer etal., 2017), cells expressing the mutant huntingtin (repeat version)protein, which aggregates in the cytoplasm and the nucleus, and causesneuronal toxicity in Huntington's disease (Zoghbi and On, 2000), wereanalyzed. It was observed herein that BRD-4780 did not reverse ordiminish the intracellular accumulation of a GFP-tagged mutant versionof huntingtin (97 polyQ) in HEK cells (FIGS. 15G and 15H). Thesefindings support the therapeutic potential of BRD-4780 for the treatmentof toxic proteinopathies caused by mutations in proteins that trafficthrough the secretory pathway.

Example 14: Pharmacokinetic Studies of BRD-4780, BRD-7709, and BRD-1365

Pharmacokinetic studies were performed to determine the oralbioavailability, plasma and tissue concentrations, and dose responseexposures of BRD-4780, BRD-7709, and BRD-1365 in 129S2 mice, 129S-ELITEmice, Sprague Dawley and CD rats (see Example 1 above for methods). Infasted male 129S2/SvPasCrl mice, a single dose of BRD-4780 wasadministered. The oral bioavailability of BRD-4780 (% F) was determinedto be 119%. In plasma protein binding studies, the estimated percentprotein bound was 29.7% (FIGS. 11A and 11B). In fed 12952/SvPasCrl mice,a single dose of BRD-4780 was administered orally. Plasma and tissuedrug concentrations of BRD-4780 were obtained for males (FIGS. 11C and11E) and females (FIGS. 11D and 11F). A comparison of the oralbioavailabilities of BRD-1365, BRD-7709 and BRD-4780 in 129S-ELITE micewas performed, as shown in FIGS. 11G-11J. A single dose of BRD-4780,BRD-1365 or BRD-7709 was administered. Dose response exposures ofBRD-4780 (FIGS. 11K and 11N), BRD-1365 (FIGS. 11L and 11O) and BRD-7709(FIGS. 11M and 11P) in 129S-ELITE mouse plasma, brain, kidney, liver andeye were also determined. All three compounds exhibited a high volume ofdistribution, with highest exposures in the eye. C_(max) exposures fordoses above 10 mg/kg were not dose proportional. AUC exposures forBRD-4780 and BRD-1365 were not dose proportional, but BRD-7709 AUCexposures were nearly dose proportional.

The oral bioavailabilities of BRD-4780, BRD-7709 and BRD-1365 were alsodetermined in Sprague Dawley rats. Plasma concentrations over time forindividual rats and the mean of 2 rats per group were plotted forBRD-4780 (FIG. 11Q), BRD-1365 (FIG. 11R) and BRD-7709 (FIG. 11S).Standard pharmacokinetic parameters were calculated (FIG. 11T). The oralbioavailability (% F) was determined to be 40.6%. for BRD-4780, 0.083,0.5, 1, 3, 6, 10, 24, 32 and 48 hours 32.7% for BRD-1365 and 56.1% forBRD-7709, with BRD-7709 showing the highest oral bioavailability, shownin FIG. 11U.

In addition, the dose-response exposure of BRD-4780, BRD-7709 andBRD-1365 were determined in male and female CD (Sprague Dawley) rats.Plasma concentrations over time for individual rats and the mean of 3rats per group were plotted for BRD-4780 in male rats (FIG. 11V) andfemale rats (FIG. 11W), for BRD-7709 in male rats (FIG. 11X) and femalerats (FIG. 11Y), and for BRD-1365 in male rats (FIG. 11Z) and femalerats (FIG. 11AA). The 24 hour and 48 hour time points for one of threemale rats in the BRD-1365 10 mg/kg group were excluded as likelytechnical outliers. Standard pharmacokinetic parameters were calculatedfor BRD-4780 (FIG. 11AB), BRD-7709 (FIG. 11AC), and FIG. 11AD). Incontrast to mice, in which AUC exposure values were higher in male micethan in female mice, AUC values were 1.4 to 2.3 fold higher in femalerats than in male rats.

Example 15: Preparation of Enantiopure Analogs of BRD-4780

BRD-4780 was prepared as a mixture of enantiomers by Diels-Alderreaction of (E)-3-methyl-1-nitrobut-1-ene, 1, and cyclopentadienefollowed by catalytic hydrogenation and HCl salt formation (scheme 1;Munk et al. WO 96/01813A1; Munk et al. J Med Chem. 39: 1193-1195). Anapproach was developed herein to prepare the individual stereoisomers,(1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-amine and(1S,2S,3S,4R)-3-isopropylbicyclo[2.2.1]heptan-2-amine and correspondingsalts, based on chiral SFC chromatography. The absolute stereochemistryof the enantiopure fractions can be assigned by formation of thecorresponding Mosher amides (Dale and Mosher. J Am Chem Soc. 95:512-519).

Most preparative scale chromatography systems rely on analyte detectionby UV-Vis absorbance (PDA detector). On the analytical scale, monitoringof the mobile-phase by mass spectroscopy is available. Potentialseparation of the (2R, 3R) and (2S, 3S) BRD-4780 was profiled across 5chiral stationary phases (OJ-H, AD-H, AS-H, IC and OD-H) using fourdifferent organic solvent systems (MeOH, MeOH+1% TFA, MeOH+0.05% Et₃Nand iPrOH) as monitored by MS detection. No separation was observed.Accordingly, a method was pursued in which detection and separation werefacilitated by incorporation of an amino protecting group that could bedetected by UV absorbance.

During the preparation of BRD-4780, the initial Diels-Alder reactiongenerated an approximately 2:1 mixture of endo and exo isomers ofracemic 5-isopropyl-6-nitrobicyclo[2.2.1]hept-2-ene, 2 (Munk et al. JMed Chem. 39: 1193-1195). According to the literature preparation of thecompound, separation of the endo and exo isomers could not be achievedat this stage and was performed following subsequent reduction to formthe racemic mixture of C2-endo and C2-exoisopropylbicyclo[2.2.1]heptan-2-amine (Id.). The ability to separate themixture of 5-isopropyl-6-nitrobicyclo[2.2.1]hept-2-ene stereoisomers wasprofiled using chiral SFC chromatography as monitored by UV-Visabsorbance (PDA detector). Separation of at least four materials wasobserved using AD-H stationary phase with iPrOH as a mobile phase (Table2, entry 10). However, this separation was not useful on the preparativescale due to peak overlap. Without wishing to be bound by theory, if anendo selective Diels-Alder reaction were developed, then separation ofthe (5R, 6R) and (5S, 6S) stereoisomers could likely be effectivedirectly on the racemic(1S,4R,5R,6R)-5-isopropyl-6-nitrobicyclo[2.2.1]hept-2-ene.

To facilitate the detection and separation of BRD-4780, a series of nineamino protected compounds were synthesized and profiled for separationacross five chiral stationary phases (OJ-H, AD-H, AS-H, IC and OD-H)using four initial solvent systems (MeOH, MeOH+1% TFA, MeOH+0.05% Et₃Nand iPrOH) (FIG. 18). The instant study included four carbamateprotecting groups (3: Cbz, 4: FMOC, 5: p-NO₂-Cbz, and 6: p-Br-Cbz), twosulfonamides (7: tosyl and 8: nosyl), the 9: N-dibenzyl, the 10:phthalimido and an acetamide formed from racemic(1S,2R,3R,4R)-3-isopropylbicyclo[2.2.1]hept-5-en-2-amine, 11, which wasprepared by selective reduction of racemic(1S,4R,5R,6R)-5-isopropyl-6-nitrobicyclo[2.2.1]hept-2-ene. Wherepromising separation was observed using one of the systems, in depthmethods of development were pursued.

The results of the instant separation study are summarized in Table 2.For descriptive purposes, “weak separation” has been defined in Table 2as two observable peaks with apparent Gaussian peak shape that do notapproach baseline separation, “moderate separation” as two observablepeaks with apparent Gaussian peak shape with near base-line separationand “well separated” as two observable peaks with apparent Gaussian peakshape that reach full baseline separation. All other cases in whichthere was no observable separation or in which the peak shape was poorlydefined have been described as “no separation.” For the well separatedexperiments, the Δ_(tr) has been reported as a relative indication ofseparation efficiency.

The tosyl protected amine, 7, and the p-NO₂-Cbz compound 5 showed thebest separation on column AD-H, thus they were optimized with othersolvents systems. As shown in Table 3, analog 7 showed better separationwhen using either methanol, basic or acidic solvents (Δt_(r)=0.78 min).Meanwhile, the p-NO₂-Cbz 5 did not show improvement in the enantiomersseparation when changing solvents, as iPrOH remained the best mobilephase (Δt_(r)=0.46 min) (FIG. 19). Thep-NO₂-Cbz 5 was used for the gramscale reaction because it was shown that this could be convenientlyremoved by catalytic hydrogenation.

Next, 5 was synthesized at gram scale in 90% yield using thecorresponding chloroformate with sodium bicarbonate in water and dioxaneat room temperature, per scheme 2 below. The enantiomers were cleanlyseparated during SFC chromatography. Full baseline separation of 25 mginjections was observed on chiralpak AD-H column (250×21 mm, 5 um, 90mL/min, 9:1 CO₂:IPA) and with high recovery (Fraction, ‘Fr’, 1: 88% oftheoretical yield, Fr2: 91% of theoretical yield). Analytical chiralHPLC analysis of the isolated fractions demonstrated a single peak andno absorption at the retention time of the other enantiomer was detected(>99% ee). Deprotection of the p-NO₂-Cbz group proceeded well usingPdCl₂ in ethyl acetate under an atmosphere of H₂ giving 51% yieldfollowing purification, salt formation and trituration in pentane.Following formation of the HCl salt, the analytical characterization wasidentical with authentic BRD-4780 other than the specific rotation whichshowed approximately equal and opposite values: Fr1: [a]²⁵ _(D)+12.0°(c=0.1, MeOH); Fr2: [a]²⁵ _(D)−13.0° (c=0.1, MeOH).

To determine the absolute configuration of BRD-4780 enantiomers,Mosher's model was employed. First, the (+/−) BRD-4780 was derivatizedwith optically-pure (S)-Mosher's acid chloride. The resultingdiastereomeric mixture of (S)-Mosher amides were analyzed by ¹H NMRspectroscopy and compared to the BRD-4780 ¹H NMR spectrum. The BRD-4780C3 proton has a chemical shift of 1.18 ppm while the C3 proton of themixture of (R)-Mosher amide diastereomers displayed two magneticallyinequivalent peaks well resolved and separated with chemical shifts of0.57 ppm and 0.52 ppm. Then, the (R)- and (S)-Mosher amides weresynthesized with both pure BRD-4780 enantiomers. For the BRD-4780enantiomer with (2R, 3R) configuration, the C3 proton of the R-Mosherdiastereoisomer is more downfield than that of the (S)-Mosherdiastereoisomer (Fr2). For the enantiomer with the absoluteconfiguration of (2S, 3S), the C3 proton of the (R)-Mosherdiastereoisomer is more upfield than the amide formed from the(S)-Mosher acid (FIGS. 20A and 20B).

The pure enantiomers were profiled in the high-content imaging assay forthe ability to reduce the amount of FS MUC1 protein in the cytoplasm(Table 1). As was the case for the racemic compound, BRD-4780, there wasno observed reduction in wild-type MUC1 levels.

TABLE 1 Bioactivity of enantiopure BRD-4780 Fr1 and Fr2 Fraction, FSMUC1 FS MUC1 Optical cytoplasm IC₅₀ cytoplasm EMax rotation (uM) (%)Fr1, (+) 0.73 −39 Fr2, (−) 0.66 −39

In summary, a method for the preparation of(1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-amine and(1S,2S,3S,4R)-3-isopropylbicyclo[2.2.1]heptan-2-amine and thecorresponding hydrochloride salts in high enantiopurity has beendeveloped, and the absolute stereo-chemistry of the individualenantiomers has been assigned using the Mosher amide method. The instantprocedure relies on derivatization of the BRD-4780 amino group, whichenabled detection and appeared to be required for efficient separationon the stationary phase identified by methods screening and development.The applicability of this approach has been demonstrated on the hundredmilligram scale, and it is projected that the instant process will beapplicable to larger scale preparative applications (Caille et al.(2010) Org Process Res Dev. 14: 133-141). The ability of these compoundsto remove FS MUC1 protein from cells has been profiled in vitro.

TABLE 2 Summary of Example 15 Compounds Col2: OJ—H Col3: AD—H Col4: AS—HCol5: IC Col6: OD—H 3-50% 3-50% 3-50% 3-50% 3-50% Entry Compounds iPrOHiPrOH iPrOH iPrOH iPrOH 1

Moderate separation Δt_(R) = 0.14 min No separation No separation Noseparation No separation 2

Weak separation Δt_(R) = 0.06 min Weak separation Δt_(R) = 0.05 min Noseparation No separation No separation 3

No separation Well separated Δt_(R) = 0.46 min No separation Noseparation Weak separation Δt_(R) = 0.34 min 4

Moderate separation Δt_(R) = 0.13 min Moderate separation Δt_(R) = 0.13min No separation No separation Moderate separation Δt_(R) = 0.21 min 5

No separation Well separated Δt_(R) = 0.27 min No separation Noseparation No separation 6

No separation No separation Moderate separation Δt_(R) = 0.15 min Noseparation No separation 7

Weak separation Δt_(R) = 0.08 min No separation No separation Noseparation No separation 8

No separation No separation No separation No separation No separation 9

No separation Weak separation Δt_(R) = 0.1 min Weak separation Δt_(R) =0.15 min Weak separation Δt_(R) = 0.15 min No separation 10

No separation Weak separation- multiple peaks from reaction mixture Noseparation No separation No separation 11

No separation No separation No separation No separation No separation

TABLE 3 Optimization of Compounds 5 and 7 with Other Solvents SystemsChiral B2:MeOH + B3:MeOH + Entry Solvents Column B1:MeOH 0.1% TFA 0.05%Et₃N B4:iPrOH 1

Col3: AD—H Moderate separation Δt_(R) = 0.14 min Moderate separationΔt_(R) = 0.13 min Moderate separation Δt_(R) = 0.13 min Well separatedΔt_(R) = 0.46 min 2

Col3: AD—H Well separated Δt_(R) = 0.76 min Well separated Δt_(R) = 0.78min Well separated Δt_(R) = 0.78 min Moderate separation Δt_(R) = 0.27min

Experimental Processes

Analytical scale SFC conditions: columns: CHIRALCEL OJ-H, AD-H, AS-H, ICand OD-H (250×4.6 mm×5 um); Flow rate: 1.5 mL/min; Mobile phases: MeOH,MeOH+1% TFA, MeOH+0.05% Et₃N and iPrOH; ABPR: 136 Bar; Column oventemp.: 45° C.

Preparative scale SFC conditions for the separation of 3: Column:CHIRALCEL OX-H (250×21 mm×5 um); Flow rate: 85 mL/min; Mobile phase:Line-A: 93% of Liq. CO₂, Line-B: 7% of 0.1% DEA in IPA: Acetonitrile(50:50); Sample injection: 10 mg; ABPR: 100 Bar; Column oven temp.:ambient. Incomplete separation.

Preparative scale SFC conditions for the separation of 5: Column:CHIRALCEL AD-H (250×21 mm×5 um); Flow rate: 90 mL/min; Mobile phase:Line-A: 90% of Liq. CO₂, Line-B: 10% IPA; Sample injection: 25 mg; ABPR:100 Bar; Column oven temp.: ambient. Full Baseline separation.

Procedures

rac-benzyl((1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-yl)carbamate, 3

To a stirred solution of rac-(1R,2R,3R,4S)-3-isopropylnorbornan-2-aminehydrochloride (37 mg, 0.196 mmol, 1.00 eq) and disodium carbonate (22mg, 0.206 mmol, 1.05 eq) in water (1 mL), at 0° C. was added slowlybenzyl carbonochloridate (0.028 mL, 0.196 mmol, 1.00 eq). After 20 minof stirring, additional water (0.5 mL) was added and the reactionmixture was stirred for another hour. After complete addition, diethylether was added and the product was extracted 3 times with ether. Thecombined organic layers were washed with HCl (1 M) and NaOH (1 M), driedwith MgSO4, filtered and concentrated. The crude residue was purifiedwith flash chromatography on silica gel (Hexane/EtOAc) to afford thedesired racemic benzyl((1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-yl)carbamate (25 mg,44% yield) as a solid.

¹H NMR (400 MHz, Chloroform-d) δ 7.36 (m, 5H), 5.11 (m, 2H), 4.79 (d,J=7.9 Hz, 1H), 3.62 (m, 1H), 2.43 (m, 1H), 2.12 (d, J=4.2 Hz, 1H),1.65-1.57 (m, 2H), 1.51 (m, 4H), 1.22 (dd, J=10.1 Hz, 2.1 Hz, 1H), 1.14(m, 1H), 0.89 (m, 6H), 0.49 (m, 1H).

¹³C NMR (101 MHz, Chloroform-d) δ 155.90, 136.81, 128.66, 128.21, 66.70,58.52, 58.42, 41.04, 39.43, 35.61, 32.24, 30.87, 21.86, 21.18, 20.23.

MS(ESI): 288.7 [M+H]+

rac-(9H-fluoren-9-yl)methyl((1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-yl)carbamate, 4

To a stirred solution ofrac-(1R,2R,4S)-3-(1-methylethyl)norbornan-2-amine hydrochloride (37 mg,0.196 mmol, 1.00 eq) in 1,4-dioxane (1 mL), was added sodium carbonate(1.0 M in water, 0.21 mL, 0.206 mmol, 1.05 eq). At 0° C., a solution of9H-fluoren-9-ylmethyl carbonochloridate (51 mg, 0.196 mmol, 1.00 eq) indioxane (0.2 ml) was added. The reaction mixture was allowed to warm upto room temperature and was stirred for overnight. Water was poured intothe reaction mixture and the product was extracted with EtOAc. Theorganic layer was dried with MgSO₄, filtered and concentrated. The cruderesidue was purified with flash chromatography (Hexane/EtOAc 0-30%) toafford the desired rac (9H-fluoren-9-yl)methyl((1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-yl)carbamate (30 mg,41% yield).

¹H NMR (400 MHz, Chloroform-d) δ 7.77 (d, J=7.5 Hz, 2H), 7.64-7.56 (m,2H), 7.45-7.36 (m, 2H), 7.32 (ddd, J=7.4, 7.4, 1.2 Hz, 2H), 4.79 (d,J=7.5 Hz, 1H), 4.44 (m, 2H), 4.23 (m, 1H), 3.59 (m, 1H), 2.42 (s, 1H),2.13 (m, 1H), 1.68-1.50 (m, 1H), 1.48-1.30 (m, 3H), 1.22 (s, 2H),0.95-0.83 (m, 6H), 0.50 (ddd, J=9.8, 5.4, 2.0 Hz, 1H).

13C NMR (101 MHz, DMSO-d6) δ 155.61, 143.93, 143.88, 140.72, 127.60,127.00, 125.25, 125.21, 120.10, 65.03, 58.12, 54.29, 46.84, 40.60,39.52, 38.78, 36.22, 35.03, 31.74, 29.94, 21.75, 20.79, 19.73.

MS(ESI): 376.5 [M+H]

rac-4-nitrobenzyl((1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-yl)carbamate, 5

To a vial containing rac-(1R,2R,3R,4S)-3-isopropylnorbornan-2-aminehydrochloride (1.00 g, 5.27 mmol, 1.00 eq) and (4-nitrophenyl)methylcarbonochloridate (1.19 g, 5.53 mmol, 1.05 eq) were added dioxane (25mL) and sodium carbonate (1.0 M in water, 5.53 mL, 5.53 mmol, 1.05 eq).The reaction mixture was stirred at room temperature for 18 hours. Waterwas poured into the reaction mixture and the product was extracted withEtOAc. The organic layer was dried with MgSO₄, filtered andconcentrated. The crude residue was purified with flash chromatography(silica gel, Hexane/EtOAc 0-40%) to afford the desired productrac-4-nitrobenzyl((1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-yl)carbamate (1.57 g,90% yield).

¹H-NMR (400 MHz, Chloroform-d) δ 8.22 (d, J=8.5 Hz, 2H), 7.50 (d, J=8.4Hz, 2H), 5.18 (m, 2H), 4.83 (d, J=7.9 Hz, 1H), 3.59 (m, 1H), 2.43 (m,1H), 2.15 (d, J=3.6 Hz, 1H), 1.60-1.52 (m, 2H), 1.49-1.33 (m, 4H),1.23-1.10 (m, 2H), 0.89 (m, 6H), 0.50 (m, 1H).

MS (ESI): 333.3 [M+H]+

rac-4-bromobenzyl((1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-yl)carbamate, 6

To a stirred solution of rac-(2R,3R)-3-isopropylnorbornan-2-aminehydrochloride (30 mg, 0.158 mmol, 1.00 eq) and disodium carbonate (34mg, 0.316 mmol, 2.00 eq) in 1,4-dioxane (1 mL) was added(4-bromophenyl)methyl carbonochloridate (24 uL, 0.158 mmol, 1.00 eq).The reaction mixture was stirred at room temperature for 24 hours. Thereaction mixture was filtered and the filtrate was concentrated. Thecrude residue was purified with flash chromatography (Hexane/EtOAc0-25%) to afford the desired rac-4-bromobenzyl((1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-yl)carbamate (9 mg,16%) as a white solid.

¹H-NMR (400 MHz, Chloroform-d) δ 7.51 (d, J=8.0 Hz, 2H), 7.24 (d, J=8.0Hz, 2H), 5.03 (m, 2H), 4.76 (d, J=7.9 Hz, 1H), 3.58 (m, 1H), 2.42 (s,1H), 2.13 (d, J=3.6 Hz, 1H), 1.64-1.58 (m, 3H), 1.49-1.32 (m, 4H), 1.19(dd, J=10.3, 2.0 Hz, 1H), 1.16-1.07 (m, 1H), 0.88 (m, 6H), 0.51-0.43 (m,1H).

¹³C NMR (101 MHz, Chloroform-d) δ 155.69, 145.43, 131.78, 129.84, 65.84,58.48, 58.45, 41.02, 39.41, 35.60, 35.41, 32.23, 30.82, 21.86, 21.17,20.21.

MS (ESI): 336.2/368.3 [M+H]+

rac-N-((1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-yl)-4-methylbenzenesulfonamide,7

To a stirred solution of rac-(2R,3R)-3-isopropylnorbornan-2-aminehydrochloride (39 mg, 0.254 mmol, 1.00 eq) in dry dichloromethane (1mL), at 0° C. and under N₂ was added triethylamine (0.071 mL, 0.509mmol, 2.00 eq) and a solution of 4-methylbenzenesulfonyl chloride (53mg, 0.280 mmol, 1.10 eq) in DCM (0.2 ml). The reaction mixture wasallowed to warm up to room temperature and was stirred for 2.5 days.Water was poured into the reaction mixture and the product was extractedwith DCM. The organic layer was dried with MgSO₄, filtered andconcentrated. The crude residue was purified with flash chromatography(silica gel, Hexane/EtOAc 0-15%) to afford the desired racN-((1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-yl)-4-methylbenzenesulfonamide(36 mg, 57% yield).

¹H NMR (400 MHz, Chloroform-d) δ 7.77 (d, J=8.2 Hz, 2H), 7.28 (d, J=8.2Hz, 2H), 4.74 (d, J=6.7 Hz, 1H), 3.13 (ddd, J=6.8, 5.3, 3.9 Hz, 1H),2.42 (s, 3H), 2.11-2.01 (m, 2H), 1.56-1.42 (m, 2H), 1.34-1.17 (m, 3H),1.10 (m, 2H), 0.82 (d, J=6.5 Hz, 3H), 0.69 (d, J=6.6 Hz, 3H), 0.51 (ddd,J=9.5, 5.1, 2.1 Hz, 1H).

¹³C NMR (101 MHz, Chloroform-d) δ 143.39, 137.88, 129.68, 127.42, 60.42,58.72, 40.78, 38.88, 35.45, 32.18, 30.59, 21.80, 21.69, 20.90, 20.00.

MS (ESI): 306.1 [M−H]−

rac-N-((1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-yl)-2-nitrobenzenesulfonamide,8

To a stirred solution ofrac-(1R,2R,3R,4S)-3-(1-methylethyl)norbornan-2-amine (30 mg, 0.196 mmol,1.00 eq) in dry dichloromethane (1 mL), at 0° C. and under N₂ was addedtriethylamine (0.055 mL, 0.391 mmol, 2.00 eq) and a solution of2-nitrobenzenesulfonyl chloride (48 mg, 0.215 mmol, 1.10 eq) in DCM (0.2ml). The reaction mixture was allowed to warm up to room temperature andwas stirred for 1 hour. Water was poured into the reaction mixture andthe product was extracted with DCM. The organic layer was dried withMgSO₄, filtered and concentrated to afford the desiredrac-N-((1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-yl)-2-nitrobenzenesulfonamide(37 mg, 56% yield).

¹H NMR (400 MHz, Chloroform-d) δ 8.19-8.12 (m, 1H), 7.88-7.81 (m, 1H),7.79-7.68 (m, 2H), 5.41 (d, J=7.0 Hz, 1H), 3.37 (ddd, J=7.0, 5.4, 4.0Hz, 1H), 2.25-2.04 (m, 2H), 1.63-1.42 (m, 2H), 1.42-1.07 (m, 6H), 0.85(d, J=6.6 Hz, 3H), 0.70 (d, J=6.6 Hz, 3H), 0.63 (ddd, J=9.7, 5.2, 2.1Hz, 1H).

MS (ESI): 337.2 [M−H]−

rac-(1R,2R,3R,4S)−N,N-dibenzyl-3-isopropylbicyclo[2.2.1]heptan-2-amine,9

To stirred a solution ofrac-(1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-amine (0.3 g, 1.960mmol, 1.0 eq) in N,N-dimethylformamide (3 mL) and potassium carbonate(0.541 g, 3.92 mmol, 2 eq) was added and reaction mass was stirred for15 min at room temperature. To this, benzyl bromide (0.268 g, 1.568mmol, 0.8 eq) was added drop wise and reaction mass was stirred at roomtemperature for 1 h. After completion of reaction, reaction mixture wasdiluted with water (10 mL) and extracted with dichloromethane (2×10 mL).The combined organic was dried over anhydrous sodium sulfate, filteredand concentrated to get crude material, which was purified using silicagel column chromatography (10% ethyl acetate in hexanes) to get (0.1 g,0.299 mmol, 15%) of the product.

MS (ESI): 334.2 [M+H]+.

rac-2-((1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-yl)isoindoline-1,3-dione,10

To a pressure vessel rac-(2R,3R)-3-isopropylbicyclo[2.2.1]heptan-2-amine(0.150 g, 0.980 mmol, 1 eq.) in N,N-dimethylacetamide (2 mL) was addedisobenzofuran-1,3-dione (0.156 g, 1.043 mmol, 1.6 eq) and the reactionmixture was stirred at 180° C. for 3 h. After completion of reaction,reaction mixture was diluted with water and extracted with ethyl acetate(3×20 mL) and the combined organic layer was dried over anhydrous sodiumsulphate, filtered and concentrated to get crude, which was purified bysilica gel column chromatography (3% ethyl acetate in hexanes) thedesired product (160 mg, 0.564 mmol, 58%).

¹H NMR (400 MHz, Chloroform-d) δ 7.86 (m, 2H), 7.74 (m, 2H), 4.13 (m,1H), 2.53-2.48 (m, 2H), 2.41 (m, 1H), 1.78-1.68 (m, 1H), 1.68-1.61 (m,2H), 1.46-1.40 (m, 2H), 1.30-1.24 (m, 2H), 0.98 (d, J=6.5 Hz, 3H), 0.76(d, J=6.6 Hz, 3H).

MS (ESI): 284.1 [M−H]⁺.

N-((3R)-3-isopropylbicyclo[2.2.1]hept-5-en-2-yl)acetamide, 11

To a stirred solution of cruderac-(1S,4R,6R)-5-isopropyl-6-nitro-bicyclo[2.2.1]hept-2-ene (endo:exoNO₂ is ˜2:1) (5.39 g, 27.6 mmol, 1.00 eq) in (1:1) mixture of methanol(50 mL) and aqueous solution of saturated ammonium formate (50 mL) wasadded lot wise zinc dust (9.02 g, 138 mmol, 5.00 eq) over a period of 10minutes at room temperature. The resulting reaction mixture was stirredat room temperature for 12 h. After completion of the reaction, reactionmass was filtered through celite pad and washed with methanol (2×30 mL).The organic layer was basified with saturated ammonium bicarbonate(150-160 mL) till pH=9-10. The resultant aqueous layer was extractedwith dichloromethane (2×90 mL), and the combined organic layers weredried over sodium sulphate, filtered through celite, and evaporated inunder vacuum at low temperature to get impure product cruderac-(3R)-3-isopropylbicyclo[2.2.1]hept-5-en-2-amine (4 g, 26.45 mmol,96%) and used in next step with our further purification. MS (ESI):152.2 [M+H]⁺

To a stirred solution of the cruderac-(3R)-3-isopropylbicyclo[2.2.1]hept-5-en-2-amine (2.00 g, 13.2 mmol,1.00 eq) and triethyl amine (4.6 mL, 33.1 mmol, 2.50 eq) in toluene (30mL) was added acetyl chloride (1.4 mL, 19.8 mmol, 1.50 eq) at 0° C. Thereaction mixture was stirred at room temperature for 12 h. Aftercompletion of reaction, the reaction was diluted by addition of water(30 mL), and the layers were separated. The aqueous layer was extractedwith dichloromethane (2×50 mL) and the combined organics were dried overanhydrous sodium sulphate, filtered through celite and evaporated toprovide crude material, which was purified by silica gel chromatography(25% ethyl acetate in hexanes) to provideN-[rac-(2R,3R)-3-isopropyl-2-bicyclo[2.2.1]hept-5-enyl]acetamide, 11(endo isomer, 0.65 g, 3.36 mmol, 25%). TheN-[rac-(2S,3S)-3-isopropyl-2-bicyclo[2.2.1]hept-5-enyl]acetamide elutedafter the endo isomer was also isolated (exo isomer, 0.55 g, 2.85 mmol,22%).

¹H NMR (400 MHz, Chloroform-d) δ 6.42 (dd, J=5.8, 3.2 Hz, 1H), 6.06 (dd,J=5.7, 2.8 Hz, 1H), 5.15 (bs, 1H), 4.16 (m, 1H), 3.00 (m, 1H), 2.73 (m,1H), 1.93 (s, 3H), 1.68 (s, 2H), 1.59-1.41 (m, 1H), 1.33-1.25 (m, 1H),0.98 (m, 6H).

MS (ESI): 194.2 [M+H]+

Representative deprotection of enantiomerically pure 5. (+) and (−)BRD-4780

To a stirred solution of PdCl₂ (250 mg, w/w) in ethyl acetate (2.5 mL)was added a solution of enantiopure 4-nitrobenzyl((1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-yl)carbamate (250 mg,0.752 mmol) in ethyl acetate (2.5 mL)) under nitrogen atmosphere. Thereaction mass was stirred at room temperature for 2 h under hydrogenpurging. The reaction mixture was filtered through celite bed and washedwith ethyl acetate (2×15 mL). The filtrate was concentrated, dissolvedin 2 mL of dichloromethane and cooled to 0° C. 4M hydrochloric acid in1,4-dioxane (0.2 mL) was added drop wise and reaction mixture wasstirred at room temperature for 15 min, concentrated and triturated withn-pentane to get (1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-aminehydrochloride (73 mg, 0.385 mmol, 51%). NMR and MS characterization wereidentical to authentic racemic AGN192403. Fraction 1: [a]²⁵ _(D)+12.0°(c=0.1, MeOH); Fraction 2: [a]²⁵ _(D)−13.0° (c=0.1, MeOH).

BRD-4780 (R) Mosher amide, Fr1.(R)-3,3,3-trifluoro-N-((1S,2S,3S,4R)-3-isopropylbicyclo[2.2.1]heptan-2-yl)-2-methoxy-2-phenylpropanamide

To a stirred solution of(1S,2S,3S,4R)-3-isopropylbicyclo[2.2.1]heptan-2-amine hydrochloride (10mg, 0.053 mmol, 1.00 eq) and N,N-diethylethanamine (22 uL, 0.158 mmol,3.00 eq) in dichloromethane (0.50 mL) was added(2S)-3,3,3-trifluoro-2-methoxy-2-phenyl-propanoyl chloride (15 mg,0.0580 mmol, 1.10 eq). The reaction mixture was stirred at roomtemperature for 1 hour. Then, the reaction mixture was concentrated andpurified with flash chromatography (silica gel, Hexane/EtOAC 0-30%) toafford the desired(R)-3,3,3-trifluoro-N-((1S,2S,3S,4R)-3-isopropylbicyclo[2.2.1]heptan-2-yl)-2-methoxy-2-phenylpropanamide(10 mg, 51% yield).

¹H NMR (400 MHz, Chloroform-d) δ 7.55-7.49 (m, 2H), 7.40-7.35 (m, 3H),6.64 (d, J=8.0 Hz, 1H), 3.83 (m, 1H), 3.45 (q, J=1.6 Hz, 3H), 2.50 (m,1H), 2.16 (d, 3.6 Hz, 1H), 1.61 (m, 1H), 1.46 (m, 3H), 1.37 (m, 1H),1.24-1.19 (m, 2H), 0.87 (d, J=6.6 Hz, 3H), 0.75 (d, J=6.6 Hz, 3H), 0.52(ddd, J=9.9, 5.2, 2.0 Hz, 1H).

13C NMR (101 MHz, Chloroform-d) δ 165.68, 132.90, 129.53, 128.53,127.75, 125.35, 122.47 58.24, 56.79, 55.26, 41.10, 39.54, 35.81, 32.09,30.74, 21.90, 21.31, 20.06.

BRD-4780-(S) Mosher amide, Fr1.(S)-3,3,3-trifluoro-N-((1S,2S,3S,4R)-3-isopropylbicyclo[2.2.1]heptan-2-yl)-2-methoxy-2-phenylpropanamide

To a stirred solution of(1S,2S,3S,4R)-3-isopropylbicyclo[2.2.1]heptan-2-amine hydrochloride (10mg, 0.0527 mmol, 1.00 eq) and N,N-diethylethanamine (22 uL, 0.158 mmol,3.00 eq) in dichloromethane (0.50 mL) was added(2R)-3,3,3-trifluoro-2-methoxy-2-phenyl-propanoyl chloride (15 mg,0.0580 mmol, 1.10 eq). The reaction mixture was stirred at roomtemperature for 1 hour. Then, the reaction mixture was concentrated andpurified with flash chromatography (silica gel, Hexane/EtOAC 0-30%) toafford the desired(R)-3,3,3-trifluoro-N-((1S,2S,3S,4R)-3-isopropylbicyclo[2.2.1]heptan-2-yl)-2-methoxy-2-phenylpropanamide(8 mg, 31% yield).

¹H NMR (400 MHz, Chloroform-d) δ 7.55-7.49 (m, 2H), 7.43-7.35 (m, 3H),6.63 (d, J=7.7 Hz, 1H), 3.83 (m, 1H), 3.44 (q, J=1.6 Hz, 3H), 2.50 (m,1H), 2.16 (d, 3.6 Hz, 1H), 1.59 (m, 2H), 1.51-1.45 (m, 1H), 1.45-1.36(m, 1H), 1.24-1.11 (m, 3H), 0.92 (d, J=6.5 Hz, 3H), 0.87 (d, J=6.6 Hz,3H), 0.58 (ddd, J=10.0, 5.3, 2.0 Hz, 1H).

¹³C NMR (101 MHz, Chloroform-d) δ 165.90, 133.10, 129.55, 128.60,127.81, 125.35, 122.47, 58.22, 56.93, 55.21, 40.71, 39.52, 35.75, 32.24,30.78, 21.94, 21.23, 20.08.

BRD-4780-(R) Mosher amide, Fr2.(R)-3,3,3-trifluoro-N-((1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-yl)-2-methoxy-2-phenylpropanamide

To a stirred solution of(1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-amine hydrochloride (12mg, 0.0527 mmol, 1.00 eq) and N,N-diethylethanamine (22 uL, 0.158 mmol,3.00 eq) in dichloromethane (0.50 mL) was added(2S)-3,3,3-trifluoro-2-methoxy-2-phenyl-propanoyl chloride (15 mg,0.0580 mmol, 1.10 eq). The reaction mixture was stirred at roomtemperature for 2 hours. Then, the reaction mixture was concentrated andpurified with flash chromatography (silica gel, Hexane/EtOAC 0-30%) toafford the desired(R)-3,3,3-trifluoro-N-((1S,2S,3S,4R)-3-isopropylbicyclo[2.2.1]heptan-2-yl)-2-methoxy-2-phenylpropanamide(14 mg, 60% yield).

¹H NMR (400 MHz, Chloroform-d) δ 7.56-7.47 (m, 2H), 7.42-7.33 (m, 3H),6.63 (d, J=7.9 Hz, 1H), 3.87-3.80 (m, 1H), 3.44 (d, J=1.6 Hz, 3H), 2.49(m, 1H), 2.16 (d, J=4.2 Hz, 1H), 1.65-1.52 (m, 2H), 1.49 (m, 1H),1.44-1.29 (m, 1H), 1.23-1.12 (m, 3H), 0.92 (d, J=6.5 Hz, 3H), 0.87 (d,J=6.6 Hz, 3H), 0.58 (ddd, J=10.0, 5.3, 1.9 Hz, 1H).

¹³C NMR (101 MHz, Chloroform-d) δ 165.89, 133.11, 129.53, 128.58,127.82, 125.36, 122.48, 58.23, 56.93, 55.20, 40.72, 39.53, 35.75, 32.24,30.78, 21.92, 21.23, 20.07.

BRD-4780-(S) Mosher amide, Fr2.(S)-3,3,3-trifluoro-N-((1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-yl)-2-methoxy-2-phenylpropanamide

To a stirred solution of(1R,2R,3R,4S)-3-isopropylbicyclo[2.2.1]heptan-2-amine hydrochloride (12mg, 0.0527 mmol, 1.00 eq) and N,N-diethylethanamine (22 uL, 0.158 mmol,3.00 eq) in dichloromethane (0.50 mL) was added(2R)-3,3,3-trifluoro-2-methoxy-2-phenyl-propanoyl chloride (15 mg,0.0580 mmol, 1.10 eq). The reaction mixture was stirred at roomtemperature for 2 hours. Then, the reaction mixture was concentrated andpurified with flash chromatography (silica gel, Hexane/EtOAC 0-30%) toafford the desired(R)-3,3,3-trifluoro-N-((1S,2S,3S,4R)-3-isopropylbicyclo[2.2.1]heptan-2-yl)-2-methoxy-2-phenylpropanamide(14 mg, 60% yield).

¹H NMR (400 MHz, Chloroform-d) δ 7.55-7.49 (m, 2H), 7.41-7.35 (m, 3H),6.64 (d, J=8.2 Hz, 1H), 3.83 (m, 1H), 3.45 (q, J=1.6 Hz, 3H), 2.50 (m,1H), 2.16 (d, J=3.8 Hz, 1H), 1.61 (m, 2H), 1.52-1.41 (m, 2H), 1.36 (m,1H), 1.22 (m, 2H), 0.87 (d, J=6.5 Hz, 3H), 0.75 (d, J=6.6 Hz, 3H), 0.52(ddd, J=9.9, 5.2, 2.0 Hz, 1H).

¹³C NMR (101 MHz, Chloroform-d) δ 165.68, 132.91, 129.52, 128.53,127.75, 125.36, 122.48, 58.25, 56.80, 55.24, 41.10, 39.55, 35.81, 32.09,30.74, 21.89, 21.31, 20.06.

Example 16: Identification of Additional TMED9-Binding Agents

A further screen for TMED9-binding agents is performed upon cellsharboring a misfolded protein of the secretory pathway (e.g., a MUC1mutant protein (e.g., a MUC1 frameshift mutant protein), a UMODpathogenic variant protein (e.g., a C126R UMOD mutant protein) and/or arhodopsin mutant protein (e.g., a P23H rhodopsin mutant protein)). Cellsharboring the misfolded protein of the secretory pathway are contactedwith a library of test compounds (e.g., small molecule library, nucleicacid library and/or other macromolecule library), and immunofluorescence(or other appropriate means of detection) is employed to identify apreferential diminishment in misfolded protein levels in the cells,relative to a corresponding wild-type form of the misfolded protein.Additional TMED9-binding agents are thereby identified.

Treatments are currently lacking for toxic proteinopathies, which affecta wide range of cell types from neurons to photoreceptors to kidneycells, and result in debilitating and often fatal diseases (Bayer,2015). New drug development to prevent or halt disease progression hasbeen challenging, due in large part to the dearth of mechanism-basedapproaches (Dubnikov et al., 2017; Dugger and Dickson, 2017). Here, themolecular mechanism underlying a rare and poorly understood autosomaldominant kidney condition, MKD, has been elucidated, initially showingthat MKD is a toxic proteinopathy. Using several models, it wasdemonstrated that MKD is caused by TMED9 cargo receptor-dependentretention of mutant MUC1-fs in the early secretory compartment. A leadmolecule, BRD-4780, and its target, TMED9, were also identified. Themolecule and the target demonstrated a heretofore unknown mechanism ofaction for the removal of mutant protein, based on binding to TMED9.Herein, answers to several important questions have been provided aboutboth MKD and, more generally, mechanisms for clearance of misfoldedsecretory proteins. The instant findings also have importantimplications for future therapeutic efforts against toxicproteinopathies.

First, certain discoveries of the instant disclosure have elucidated thecellular mechanism by which the toxic proteinopathy MKD begins, with theaccumulation of a mutant neo-protein. The results obtained show thataccumulation of the protein alone is not immediately toxic, owing to theactivation of the cytoprotective ATF6 branch of the UPR, and thatadditional stress signaling likely activates the pro-apoptotic branchesof the UPR, ultimately leading to epithelial cell injury. This mayexplain the late onset of kidney failure in MKD patients (Bleyer et al.,2017), which is mirrored by the late onset of histologic changes in thekidneys of heterozygous knock-in mice. Without wishing to be bound bytheory, it is speculated that while MKD patients accumulate MUC1-fs inkidney tubular epithelial cells throughout life, additional insults(such as exposure to nephrotoxins, inflammation or infections) and thegeneral decline in UPR homeostasis that accompanies normal aging (Klaipset al., 2017) may ultimately lead to kidney failure.

In addition to revealing the molecular mechanism of MKD, the instantdisclosure has identified a lead compound, BRD-4780, that is likelycapable of clearing not only MUC1-fs, but also other misfolded proteinssuch as UMOD (C126R) and rhodopsin (P23H). In contrast, BRD-4780 had noeffect on mutant huntingtin, which accumulates in the cytoplasm andnucleus. This underscores the specificity of BRD-4780 for misfoldedproteins retained in the early secretory pathway. It is estimated thatmore than 20 known proteinopathies are associated with misfoldedproteins trapped in compartments between the ER and Golgi apparatus(Dubnikov et al., 2017; Dugger and Dickson, 2017). Thus, BRD-4780 likelyprovides a therapeutic lead for multiple proteinopathies associated withmutant protein accumulation.

In addition to identifying a lead compound, the cargo receptor TMED9(also known as p25 and p24α2; (Gomez-Navarro and Miller, 2016)) wasdiscovered to be a molecular target for BRD-4780, thereby uncovering apreviously unknown cell biological mechanism for misfolded protein cargoentrapment. Cargo receptors are proteins that span the membrane andphysically link cargo with vesicle coat subunits to efficiently andselectively recruit soluble proteins to the emerging vesicles (Barloweand Helenius, 2016; Geva and Schuldiner, 2014). MUC1-fs, trapped inTMED9 cargo receptor-enriched vesicles between the cis-Golgi and the ER,was identified herein as released by the action of BRD-4780, and wasthus allowed to traffic through the secretory pathway into endosomes andfinally into lysosomes, where it could be degraded. Precisely howBRD-4780 binding to TMED9 results in the re-routing of MUC1-fs into thelysosome remains unclear. Without wishing to be bound by theory, ifTMED9 receptors directly bind MUC1-fs cargo, BRD-4780 (and potentiallyother TMED9-binding agents) may work by blocking (either competitivelyor non-competitively) MUC1-fs binding to its TMED9 receptor.Alternatively, BRD-4780 (and potentially other TMED9-binding agents) mayblock TMED9 interactions with other coatomer or integral vesicularproteins, thereby indirectly promoting MUC1-fs anterograde trafficking.The detailed molecular interactions between BRD-4780 (and potentiallyother TMED9-binding agents), TMED9 and MUC1-fs (or other misfoldedprotein cargoes) in the early secretory pathway are currently beingexamined.

The instant disclosure has revealed a new strategy of identifying cargoreceptors that retain misfolded secretory proteins and producingcompounds that promote their release and anterograde trafficking to thelysosome as a therapeutic approach to toxic proteinopathies.

BRD-4780 is a promising therapeutic lead. While careful toxicologystudies are needed, there are several pieces of evidence that arereassuring about the safety of the compound: (i) in in vitro studies,BRD-4780 showed no overt toxicity at any concentration tested, and infact, it rescued cells from THP-induced cell death, and (ii) in in vivoexperiments, BRD-4780 was well tolerated at several doses up to 50 mg/kgwith no overt toxicity. Additionally, BRD-4780 has excellent drug-likeproperties including excellent solubility, good microsomal and plasmastability, low protein binding and excellent oral bioavailability (FIG.16). Therefore, this lead compound holds significant potential for itssuccessful development into a therapy.

In summary, the molecular mechanism underlying MKD has been identifiedherein, and a small molecule possessing exciting and promising potentialas a therapeutic lead for a class of difficult-to-treat diseases with noavailable treatments has also been discovered.

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All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe disclosure pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentdisclosure is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the disclosure. Changes therein and other uses willoccur to those skilled in the art, which are encompassed within thespirit of the disclosure, are defined by the scope of the claims.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups or other grouping of alternatives, thoseskilled in the art will recognize that the disclosure is also therebydescribed in terms of any individual member or subgroup of members ofthe Markush group or other group.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the disclosure (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better Illumina®te thedisclosure and does not pose a limitation on the scope of the disclosureunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the disclosure.

Embodiments of this disclosure are described herein, including the bestmode known to the inventors for carrying out the disclosed invention.Variations of those embodiments may become apparent to those of ordinaryskill in the art upon reading the foregoing description.

The disclosure illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present disclosure provides preferred embodiments, optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis disclosure as defined by the description and the appended claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentdisclosure and the following claims. The inventors expect skilledartisans to employ such variations as appropriate, and the inventorsintend for the disclosure to be practiced otherwise than as specificallydescribed herein. Accordingly, this disclosure includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the disclosure unless otherwise indicatedherein or otherwise clearly contradicted by context. Those skilled inthe art will recognize, or be able to ascertain using no more thanroutine experimentation, many equivalents to the specific embodiments ofthe disclosure described herein. Such equivalents are intended to beencompassed by the following claims.

We claim:
 1. A method of treating MUC1-associated kidney disease in asubject, the method comprising administering to the subject2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane or a pharmaceuticallyacceptable salt thereof in an amount effective to treat MUC1-associatedkidney disease, thereby treating MUC1-associated kidney disease in thesubject.
 2. The method of claim 1, wherein the2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane is racemic (±)2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane.
 3. The method ofclaim 1, wherein the 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptaneis (+) 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane.
 4. The methodof claim 1, wherein the2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane is (−)2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane.
 5. The method ofclaim 1, wherein the 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptaneis a hydrochloride salt of2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane.
 6. A method forselecting a composition comprising2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane for administration toa subject for treating MUC1-associated kidney disease in the subject,the method comprising: (a) identifying the subject as havingMUC1-associated kidney disease; and (b) selecting a compositioncomprising 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane foradministration to the subject in an amount effective to treatMUC1-associated kidney disease, thereby selecting the compositioncomprising 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane foradministration to the subject for treating MUC1-associated kidneydisease in the subject.
 7. The method of claim 6, wherein step (a)comprises identifying the presence in the subject of a mutation in MUC1.8. The method of claim 6, wherein the subject has one or more of thefollowing: end-stage renal disease, urinalysis revealing minimal proteinand no blood, slowly progressive kidney failure, hyperglycemia and/orgout.
 9. The method of claim 6, wherein the subject has been identifiedto be in need of dialysis or kidney transplantation.
 10. The method ofclaim 6, wherein step (a) comprises identifying the presence in thesubject of a MUC1 frameshift mutation.
 11. The method of claim 6,further comprising: (c) administering the selected2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane to the subject. 12.The method of claim 6, wherein identifying step (a) comprises use of akit consisting essentially of (i) an oligonucleotide for detection of aMUC1 frameshift mutant or (ii) an antibody capable of binding a MUC1frameshift mutant protein, and instructions for its use.
 13. The methodof claim 6, wherein the subject is human.
 14. A method for treatingMUC1-associated kidney disease in a subject, the method comprising:identifying a subject as having MUC1-associated kidney disease; andadministering 2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane or apharmaceutically acceptable salt thereof, to the subject in an amounteffective to cause reduction or improvement of a symptom ofMUC1-associated kidney disease in the subject, thereby treatingMUC1-associated kidney disease in the subject.
 15. The method of claim14, wherein said compound causes release of MUC1, from an earlysecretory compartment.
 16. The method of claim 14, wherein the subjecthas a mutation in MUC1.
 17. The method of claim 14, wherein the symptomof MUC1-associated kidney disease is selected from the group consistingof slowly progressive tubulointerstitial disease, end-stage renaldisease, and a need for dialysis or kidney transplantation.
 18. Themethod of claim 14, wherein the subject has a MUC1 frameshift mutation.19. The method of claim 14, wherein the 2endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane or a pharmaceuticallyacceptable salt thereof is administered to the subject via the oralroute.
 20. The method of claim 14, wherein the2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane or pharmaceuticallyacceptable salt thereof comprises a pharmaceutically-acceptable carrieror excipient.